Drug resistance in Giardia: Mechanisms and alternative treatments for Giardiasis

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Drug resistance in Giardia: Mechanisms and alternative treatments for Giardiasis € ello-Garcíaa, David Leitschb, Tina Skinner-Adamsc, Raúl Argu M. Guadalupe Ortega-Pierresa,∗ a

Departamento de Genetica y Biologı´a Molecular, Centro de Investigacio´n y de Estudios Avanzados del Instituto Politecnico Nacional, Ciudad de Mexico, Mexico Institute for Specific Prophylaxis and Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria c Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD, Australia *Corresponding author: e-mail address: [email protected] b

Contents 1. Introduction 2. Current treatment of Giardiasis 2.1 Drug repertoire. Mechanisms and efficacy 2.2 Refractory giardiasis and treatment failures 3. Mechanisms of drug resistance in Giardia 3.1 5-Nitroheterocyclic drugs 3.2 Benzimidazoles 4. Drugs under investigation for repurposing in giardiasis 4.1 Anti-obesity agents 4.2 Anti-rheumatic agents 4.3 Anticancer agents 4.4 Proton pump inhibitors 4.5 Inhibitors of ethanol metabolism 4.6 Beta-blockers 5. Strategies for the discovery and development of new anti-Giardia agents 5.1 Next generation synthetic compounds 5.2 Phenotypic drug screening 5.3 Target function-based drug discovery 5.4 Natural product drug discovery 6. Conclusions and perspectives Acknowledgements References

Advances in Parasitology ISSN 0065-308X https://doi.org/10.1016/bs.apar.2019.11.003

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Abstract The use of chemotherapeutic drugs is the main resource against clinical giardiasis due to the lack of approved vaccines. Resistance of G. duodenalis to the most used drugs to treat giardiasis, metronidazole and albendazole, is a clinical issue of growing concern and yet unknown impact, respectively. In the search of new drugs, the completion of the Giardia genome project and the use of biochemical, molecular and bioinformatics tools allowed the identification of ligands/inhibitors for about one tenth of
150 potential drug targets in this parasite. Further, the synthesis of second generation nitroimidazoles and benzimidazoles along with high-throughput technologies have allowed not only to define overall mechanisms of resistance to metronidazole but to screen libraries of repurposed drugs and new pharmacophores, thereby increasing the known arsenal of anti-giardial compounds to some hundreds, with most demonstrating activity against metronidazole or albendazole-resistant Giardia. In particular, cysteine-modifying agents which include omeprazole, disulfiram, allicin and auranofin outstand due to their pleiotropic activity based on the extensive repertoire of thiol-containing proteins and the microaerophilic metabolism of this parasite. Other promising agents derived from higher organisms including phytochemicals, lactoferrin and propolis as well as probiotic bacteria/fungi have also demonstrated significant potential for therapeutic and prophylactic purposes in giardiasis. In this context the present chapter offers a comprehensive review of the current knowledge, including commonly prescribed drugs, causes of therapeutic failures, drug resistance mechanisms, strategies for the discovery of new agents and alternative drug therapies.

1. Introduction Giardia duodenalis (syn: G. lamblia and G. intestinalis), an enteric protozoan parasite, is one of the most important intestinal parasites of humans and also infects domestic and wildlife animals. It causes giardiasis which is an infection of public health importance worldwide (Adam, 2001; Anim-Baidoo et al., 2016). The parasite is commonly acquired from contaminated freshwater and public water supplies. Giardia infections have high levels of morbidity in tropical countries and can also be an epidemic problem in developed countries (Kulakova et al., 2014; Oberhuber et al., 1997). Giardiasis has an estimated prevalence of 280 million cases worldwide annually. In regions where giardiasis is endemic and where environmental contamination is high the infection rates vary from 2% to 30%. In developed countries its frequency is in the range of in 2–7% (Gardner and Hill, 2001; Kulakova et al., 2014; Laupland and Church, 2005; Yoder and Beach, 2007). Children are the most affected population (Kosek et al., 2003; Savioli et al., 2006) and can be chronically infected with Giardia. They may present with malnutrition,

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failure to thrive and deficient cognitive function (Guerrant et al., 2013; Kosek et al., 2003; Savioli et al., 2006). Immunocompromised patients are also vulnerable to infection by Giardia (Adamu et al., 2013). In Asia, Africa, and Latin America, about 500,000 new giardiasis cases are reported each year (Debnath et al., 2014). Due to the impact of this disease in regions with poor socioeconomic status and developing countries, the World Health Organization has included giardiasis in its Neglected Diseases Initiative (Savioli et al., 2006). Giardia infections frequently occur without symptoms and are often selflimiting (Sullivan et al., 1992; WHO, 1995). Symptomatic acute cases, however, present severe gastrointestinal symptoms which include watery diarrhoea, epigastric pain, nausea and vomiting; chronic infections can progress to malabsorption syndrome with important weight loss, malnutrition, and failure to thrive in the paediatric population. Post-infection symptoms such as lactose intolerance (Farthing, 1997) or irritable bowel syndrome (PI-IBS) have also been reported in patients infected with Giardia (Halliez and Buret, 2013). Besides sanitation and instructional campaigns, there are no prophylactic biological methods against Giardia infections available so control is reliant on chemotherapy (Granados et al., 2012). Current pharmacological therapies for giardiasis include derivatives of the nitroimidazole family such as metronidazole, tinidazole, ornidazole and secnidazole and derivatives of the benzimidazole group including albendazole and mebendazole (Canete et al., 2006b; Escobedo et al., 2008; Kulakova et al., 2014; Lemee et al., 2000). In addition, nitazoxanide, furazolidone, quinacrine, chloroquine and paromomycin can be used as alternative treatments (Leitsch, 2015). All these agents have distinct modes of action involving a variety of targets and downstream mechanisms affecting cellular processes in trophozoites, such as proliferation and conversion into infective cysts. In the following sections a description of these drug treatments are given.

2. Current treatment of Giardiasis 2.1 Drug repertoire. Mechanisms and efficacy Metronidazole [1-(-hydroxyethyl)-2-methyl-5 nitroimidazole; MTZ] was one of the first drugs used against giardiasis, and it has been used since 1959. Nowadays, MTZ is the most frequently used drug to treat giardiasis worldwide. MTZ (Flagyl®) is given in doses ranging from 15 mg/kg/day to a maximum of 750 mg/day orally in three doses for 5–10 days (Escobedo and Cimerman, 2007). Its efficacy rate ranges from 60% to 100% and it is considered the first choice in the treatment of giardiasis since its

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commercialization (Bassily et al., 1970; Escobedo and Cimerman, 2007; Gardner and Hill, 2001). MTZ is a pro-drug that needs to be reduced at its nitro group to be cytotoxic. When the drug is inside the trophozoite, electron transport proteins from the parasite (ferredoxins) donate electrons to the nitro group of the drug (Samuelson, 1999; Upcroft and Upcroft, 1998). This drug takes advantage of anaerobic metabolic pathways present in Giardia, producing severe oxidative stress by binding to free thiol groups or proteins creating cysteine adducts, it also affects DNA by creating double strand rupture (Leitsch et al., 2012; Ordonez-Quiroz et al., 2018; Uzlikova and Nohynkova, 2014). The activation of MTZ is mediated by nitroreductase-1 (NRT-1) and by the reduction pathway mediated by Ferredoxin (Fd), which in turn is reduced by Pyruvate:Ferredoxin Oxidoreductase (PFOR). This last enzyme, transfers electrons produced during oxidative decarboxylation from pyruvate to ferredoxin. Thioredoxin reductase (TrxR) is another enzyme that directly reduces MTZ (Ansell et al., 2015; Leitsch et al., 2011). The reduction can also produce free radicals that react with essential cellular components (Upcroft and Upcroft, 1998). The reduction of MTZ generates a gradient which allows intracellular transport of the drug. This process leads to DNA damage with loss of the helical structure, deterioration of its functions and consequent trophozoite death (Gillis and Wiseman, 1996; Ordonez-Quiroz et al., 2018; Uzlikova and Nohynkova, 2014). Moreover, MTZ inhibits parasite respiration (Farthing, 1992). In patients MTZ causes some adverse effects like headache, metallic taste, darkened urine, dizziness and nausea. Less frequent effects include pancreatitis, toxicity in the central nervous system, reversible neutropenia and outlying neuropathy may also occur. Although MTZ is used as the first line of defence against various parasitic protozoa, the United States Department of Health has identified it as a carcinogenic agent given that it causes tumours in mice at high doses, but no direct linkage to cancer in humans has been found (U.S. Department of Health and Human Services, 2016). The 5-nitroimidazoles, tinidazole, secnidazole and ornidazole are also frequently used for giardiasis treatment. These drugs have a much longer half-life than metronidazole, fewer side effects and shorter treatment courses rendering them more suitable for single-dose therapies (Rossignol et al., 1984). The way in which these drugs affect the parasite is very similar to that of metronidazole (Leitsch et al., 2012). Tinidazole (Tindamax, Fasigyn, Simplotan, Sporinex) is an effective treatment against Giardia and other anaerobic microorganisms and it is given at doses of 50 mg/kg maximum 2 g orally in a single dose. The efficacy of this drug, as determined by parasite

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load, ranges from 72% to 100% (Bassily et al., 1987; Canete et al., 2006b; Pasupuleti et al., 2014). It has demonstrated a 90% cure rate when used as a single dose of 1.5 g ( Jokipii and Jokipii, 1982) and is also generally better tolerated than MTZ ( Jokipii and Jokipii, 1980). Secnidazole has also demonstrated good outcomes in clinical studies. However, its absorption is very slow resulting in longer parasite exposure and improved in situ action against parasites (Di Prisco et al., 2000). Moreover, this drug has a long half-life which allows the possibility of prescribing a single dose schedule that is important for patient compliance. The recommended dosing for adults is 2 g and 30 mg/kg in paediatric patients as a single dose. At these doses cure rate are in the range of 80–98% (Canete et al., 2006a; Di Prisco et al., 2000). Side effects of secnidazole include nausea, vomiting, and bitter taste. A good alternative in the treatment of giardiasis is ornidazole (Xynor). In its mechanism of action ferredoxin or flavodoxin transporters are implicated, resulting into amino toxic compounds which inhibit DNA synthesis. Ornidazole is given orally in a single dose of 20 to 40 mg/kg (maximum 2 g) and shows and an efficacy ranging from 90% to 100% ( Jokipii and Jokipii, 1982; Sabchareon et al., 1980). Nitazoxanide (NTZ) is a nitrothiazole pro-drug that also needs to be reduced at its nitro group to be active. Its mode of action includes the inhibition of PFOR and other enzymes such as NRT-1 and quinone reductase, which compromises cell integrity (Hoffman et al., 2007; Muller et al., 2007b). It has an effectiveness of 71–80% and has been reported to have high efficacy against MTZ resistant G. duodenalis isolates (Rossignol et al., 2010). Nitazoxanide (Alina, Allpar, Adonid, Annita, Daxon, Dexidex, Nizonide) is given at a dose of 7.5 mg/kg orally twice a day for 3 days. Albendazole (ABZ) is the treatment of choice for wide variety of parasite infections, including G. duodenalis. ABZ (Albenza, Valbazen, Zentel) is given in oral doses of 10–15 mg/kg (maximum 400 mg) once daily for 5 days. ABZ is one of the four anthelmintic drugs found in the model list of essential medicines of the World Health Organization (Leitsch, 2015). In human liver, the biotransformation of ABZ is mediated by cytochromes P450 (CYP450) and flavin-dependent monooxygenase (FMO) (Rawden et al., 2000). Albendazole sulfoxide (ABZSO) is the metabolite with therapeutic activity and there are two enantiomeric forms [() ABZSO and (+) ABZSO] with a 20:80 ratio in the blood plasma (Dayan, 2003). The route of action of the benzimidazoles family including ABZ is the selective binding to β-tubulin and the αβ-tubulin heterodimer in three residues (Tyr167, Ala198 and Tyr200), preventing polymerization and microtubule assembly, mobility

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and the transport of molecules (Kwa et al., 1995). Recently it has been reported that ABZ also induces oxidative stress, which suggests that this may be a possible alternative mechanism of toxicity (Dimitrijevic et al., 2012; Locatelli et al., 2004; Martinez-Espinosa et al., 2015). In mammals, the biotransformation of ABZ is mediated by the CYP450 system, mainly by the CYP3A4 isoform. This system consists of the CYP450 protein and two electron donor proteins, the NADPH: Cytochrome P450 oxidoreductase (CPR or CYPOR) and the cytochrome B5 (cytB5) (Iyanagi et al., 2012; Pandey and Fluck, 2013; Stiborova et al., 2016). The first electron is transferred by CYPOR to the iron of the heme group. This is reduced from ferric to ferrous (Fe-III to Fe-II), allowing the binding of molecular oxygen (O2), upon receiving the second electron by CYPOR or cytB5 promoting the excision of the O–O binding. This results in the activation of CYP450 reactive intermediate protein with monooxygenase capacity (Basudhar et al., 2015; Torres Pazmino et al., 2010). Although it is known that trophozoites are able to convert ABZ into its metabolites (Arguello-Garcia et al., 2015; Oxberry et al., 2001), it has not been determined which protein carries out this function. Due to its likely implication in ABZ resistance, the identification of this molecule, is highly desirable. Mebendazole is another benzimidazole drug that has been used to treat patients with giardiasis with varying efficacy. While several studies reported a high Giardia eradication with this drug (al-Waili et al., 1988; Vivancos et al., 2018), others showed a failure to clear the parasite in treated patients (Gascon et al., 1989). The binding of the drug to the parasite, like the binding of other benzimidazoles, causes inhibition of cytoskeleton polymerization and an important decrease of glucose uptake. The current mebendazole (Vermox) regimen for the treatment of giardiasis is 200–400 mg of mebendazole once daily for 5 days. Paromomycin (PMC) is an aminoglycoside that inhibits the synthesis of G. duodenalis proteins by interaction with the 30S and 50S ribosomal subunits resulting in poor accommodation of mRNA codons within the ribosome. It has anti-giardial activity in vitro (Edlind, 1989) and in vivo (Geurden et al., 2006) but its activity is lower than that of nitroimidazoles, quinacrine and furazolidone (Vivancos et al., 2018). Its therapeutic efficacy ranges from 55% to 90%. Paromomycin is considered a drug of choice for Giardia infections in pregnant women because it is poorly absorbed in the intestine and excreted without being metabolized (Leitsch, 2015). The recommended dose is 500 mg three times per day for 10 days in adults and 25–30 mg/kg/day (divided into three doses) in children.

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Furazolidone is a nitrofuran and, as with 5-nitroimidazoles, its activation occurs by reduction of its nitro group which is more efficient in microaerophilic parasites (Brown et al., 1996). Although its mechanism of action is not well-known, it has been reported that the reduction products from this drug involve NADH interference with some parasite molecules, including DNA. Furazolidone is well absorbed after oral administration and it is metabolized in tissues; however it effectiveness against Giardia is variable and depends somehow on patient age (Leitsch, 2015). It may cause nausea, vomiting, headache, malaise, hypersensitivity reactions, hypotension, rash and urticaria. The prescribed drug therapy is 400 mg three times a day for 7–10 days (Leitsch, 2015).

2.2 Refractory giardiasis and treatment failures Most studies of chemotherapy in clinical giardiasis do not resolve 100% of the infections, implying that treatment failure always needs to be considered. Chemotherapy failures have been observed with all of the common anti-Giardia agents including metronidazole, quinacrine, furazolidone, and albendazole. These can result from reinfection, inadequate (i.e. suboptimal) drug dose, immunosuppression, drug resistance and eventual Giardia sequestration in the gallbladder or the pancreatic duct (Lalle and Hanevik, 2018; Nash, 2001). In endemic areas reinfection is common due to high environmental contamination by infective cysts in combination with poor sanitation and hygiene. The presence of immunosuppression or reinfection is usually diagnosed in the clinic and in some cases of immunosuppressed patients, who are abnormally susceptible to giardiasis, infections are often difficult to cure. Therefore, it is important for the clinicians in cases with recurrence of symptoms after therapy to differentiate between drug resistance and reinfection. In this context is important to document truly persistent infection by diagnostic tests in stool samples or in serum samples for Giardia antigen detection (Gardner and Hill, 2001). Human giardiasis has been mainly treated with MTZ for the last 60 years, but the efficacy of this drug against Giardia is today has been compromised by MTZ resistance. Indeed, an increasing number of cases refractory to MTZ treatment have been reported in low Giardia prevalence settings. In this context, a study from Spain reported that in a group of 170 patients, treatment regimens containing one or more nitroimidazoles failed to cure 5.8% of the cases (Lopez-Velez et al., 2010). In a study carried out in a specialized

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Tropical Diseases Unit at Barcelona, Spain, patients with chronic giardiasis were prospectively analysed, and around 20% were found to be refractory to the treatment with tinidazole or MTZ (Munoz Gutierrez et al., 2013). Also this study confirmed that refractory infections originating from Asia were more prevalent (70%) than infections originating from elsewhere. Indeed, large proportions of nitroimidazole treatment failure cases came from Asia, primarily India, or the Mediterranean. Further in another study, performed in the Czech Republic it was reported that 19% of the 47 isolates genotyped from patients with giardiasis were from clinically resistant to MTZ (Lecova et al., 2018). These treatment failures will be addressed in more detail in Section 3. Treatment failures have also been reported with ABZ administered alone or in combination with MTZ (Abboud et al., 2001; Brasseur and Favennec, 1995). In addition, although nitazoxanide resistance has not been observed in the clinic, it can be readily induced in the laboratory (Muller et al., 2007b). While limited data on quinacrine resistance in G. duodenalis are available, resistance to quinacrine has also been induced in vitro (Upcroft et al., 1996a). As a general practise, clinically resistant strains have been treated with longer repeat courses or higher doses of the original agent or switching to another anti-giardial compound with a different mode of action, or combination therapy regimens of MTZ and ABZ or quinacrine may be also the strategy to avoid potential cross-resistance (Craft et al., 1981; Escobedo et al., 2016; Garg, 1972). Most of the refractory cases can be ultimately cured with available anti-giardial drugs as has been shown in a case study during an outbreak of giardiasis in Norway where a combination of drugs was used (Morch et al., 2008).

3. Mechanisms of drug resistance in Giardia 3.1 5-Nitroheterocyclic drugs 3.1.1 The phenomenon of nitroheterocycle resistance Resistance to nitroheterocyclic drugs has been observed in Giardia with all relevant drugs from this class, including 5-nitroimidazoles such as MTZ, the nitrofuran furazolidone, and the 5-nitrothiazolide nitazoxanide. Although MTZ has been in use for 60 years (reviewed in Leitsch, 2019) it has remained a mainstay in the treatment of microaerophilic and anaerobic pathogens in general. However, unsuccessful treatment of giardiasis with MTZ as a first-line monotherapy has become a growing concern in the last 15 years

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(Lalle and Hanevik, 2018). As described above, in a worrying report from the Hospital for Tropical Diseases in London, a steep increase in the number of MTZ-refractory patients from 15% to more than 40% of all cases was reported for the short period of 2008–13 (Nabarro et al., 2015). More than two thirds of these refractory cases had travelled to India. In India itself, a steep increase of refractory cases has also been reported (Yadav et al., 2014). Nitazoxanide and furazolidone are administered as first-line drugs by the way of exception only, the former exclusively in paediatric cases, so that reports on resistance are comparably scarce. It has been observed, however, that patients refractory to MTZ are also difficult to treat with other drugs, including nitazoxanide and ABZ (Meltzer et al., 2014). Isolation of MTZ-resistant Giardia from patients has only clearly been documented once (Lemee et al., 2000). In this study, Giardia cell lines from refractory patients proved to be MTZ-resistant in a mouse model. However, in other cases (Smith et al., 1982), Giardia isolates from refractory patients proved to be susceptible to MTZ when tested in vitro. The reason for this has remained unclear but assay conditions for resistance testing could play a role. Clinical MTZ resistance in T. vaginalis, for example, only manifests in the presence of oxygen which leads to deactivation of MTZ and other 5-nitroimidazoles (reviewed in Leitsch, 2019). Accordingly, clinical MTZresistant T. vaginalis strains have impaired oxygen scavenging pathways (Leitsch et al., 2014; Yarlett et al., 1986), leading to higher intracellular oxygen concentrations and, consequently, to more pronounced deactivation of 5-nitroimidazoles. As Giardia inhabits the small intestine, which is fairly well oxygenated as compared to the large intestine (Mastronicola et al., 2011), similar mechanisms could play a role in that location as well. Giardia, however, seems to be more vulnerable to oxygen than T. vaginalis (Gillin and Reiner, 1982) which complicates drug susceptibility testing considerably. Recent data also suggest that growth media constituents such as cysteine and bile can have a strong distorting effect on the test outcomes (Leitsch, 2017). These issues need be addressed to derive a better understanding of clinical MTZ resistance in Giardia. 3.1.2 Nitroheterocycle resistance induced in the laboratory Due to the lack of clinical MTZ-resistant strains available for drug resistance studies, research has concentrated on Giardia cell lines with resistance induced in the laboratory. Induction of resistance to MTZ, nitazoxanide and other nitroheterocycles is fairly simple and can be achieved by exposure to ever increasing, initially sub-lethal doses of the drug (Boreham et al., 1988;

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Townson et al., 1992) or, alternatively, by direct mutagenesis with UV-light (Townson et al., 1992). Most of the better studied resistant cell lines (Table 1) were generated at the Queensland Institute of Medical Research (QIMR, now QIMR Berghofer Medical Research Institute), namely BRIS/83/ HEPU/106-2ID10 (106-2ID10), BRIS/83/HEPU/713-M3 (713-M3), and WB-M3, with BRIS/83/HEPU/106 (106), BRIS/83/HEPU/713 Table 1 Nitroheterocycle resistant cell lines. Factors Strains

PFOR/Fd

NR1

NR2

TrxR

106-2ID10 Decreased activity, expression not affected (Ansell et al., 2017; Leitsch et al., 2011; Emery et al., 2018)

Downregulated (Ansell et al., 2017; Emery et al., 2018; Muller et al., 2019)

Not found in data set (Ansell et al., 2017; Emery et al., 2018; Muller et al., 2019)

Upregulated (Emery et al., 2018; Muller et al., 2019)

713-M3

Activity modestly decreased, expression hardly affected (Ansell et al., 2017; Emery et al., 2018; Leitsch et al., 2011; Muller et al., 2019)

Downregulated (Ansell et al., 2017; Emery et al., 2018) Not downregulated (Muller et al., 2019)

Not found in data set (Ansell et al., 2017; Emery et al., 2018; Muller et al., 2019)

Unregulated (Ansell et al., 2017; Muller et al., 2019)

WB-M3

Downregulated (Ansell et al., 2017; Emery et al., 2018; Muller et al., 2019)

Downregulated (Ansell et al., 2017; Emery et al., 2018)

Not found in Downregulated data set (Ansell (Emery et al., et al., 2017; 2018) Emery et al., 2018)

C4

Expression not Downregulated Not found in affected (Muller (Muller et al., the data set et al., 2007a) 2007a, 2019 (Muller et al., 2019)

Upregulated (Muller et al., 2019)

Expression of factors in metronidazole-resistant Giardia strains which activate (PFOR/Fd, NR1, TrxR) and deactivate (NR2) metronidazole.

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(713) and WB as the respective MTZ-sensitive parental cell lines. Cell line C4 (Muller et al., 2007a), derived from strain WB C6, is another well studied resistant cell line which displays cross resistance to nitazoxanide and MTZ. The exact degree of resistance documented for these cell lines varies between laboratories, but growth in the presence of the 10 to 100-fold the concentration of the respective drug, as compared to the drug sensitive parent cell line, is firmly supported by experimental evidence. The resistance trait is stable even if resistant cell lines are cultured without drug for extended periods of time (Emery et al., 2018). However, characteristics of laboratory-derived cell lines do not necessarily reflect the situation in vivo, including important parameters such as infectivity. This is exemplified by the impaired or even abolished capability, respectively, of 106-2ID10 and 713-M to establish infections in suckling mice (Tejman-Yarden et al., 2011). In the last 10–15 years, the field of drug research in Giardia has gained considerable momentum with important contributions made on the genetic and physiological backgrounds of resistance to nitroheterocycles. These studies were facilitated by new and powerful research tools such as quantitative high-throughput proteomics (Emery et al., 2018; Muller et al., 2019), RNAseq (Ansell et al., 2017) and Illumina whole genome sequencing (Saghaug et al., 2019). Most of the studies published, however, have to be appreciated under the caveat that exclusively Giardia assemblage A1 isolates have been used so far, although assemblages A2 and B more often cause disease in humans (Saghaug et al., 2019) and, therefore, are more often associated with treatment failure. Moreover, a recent study showed that genetic variability is higher in assemblage B isolates which might have repercussions on the emergence of resistance (Saghaug et al., 2019). Despite this limited genetic scope multiple resistance mechanisms have been discovered, many of which seem to be unique to single resistant cell lines. As a result of this complexity, our understanding of resistance to nitroheterocycles in Giardia has remained incomplete. In addition, it has also remained incomplete because no furazolidone-resistant Giardia cell lines are available, resulting in very limited knowledge about resistance to this drug. The only established notion with regard to furazolidone resistance is that it coincides with resistance to the unrelated drug quinacrine (Upcroft et al., 1996a). 3.1.3 Factors of nitroheterocycle resistance Two broad categories of drug resistance mechanisms can be defined: passive and active (Ansell et al., 2017). In the context of nitroheterocycles the former category includes reduced expression or even genetic loss of enzymes which

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reduce the nitro group of the drug and thereby prevent activation and the production of toxic intermediates. The occurrence of impaired oxygen scavenging pathways, leading to higher intracellular oxygen levels and, consequently, impaired activation of nitroheterocycles, constitutes a passive resistance mechanism, as demonstrated in clinical MTZ-resistant T. vaginalis isolates (Leitsch et al., 2014). Active mechanisms involve either drug removal (e.g., efflux pumps) or deactivation. Deactivation of nitroheterocycles can be brought about by transfer of a sufficient number of electrons, i.e., six, to the nitro group to form a relatively inert and non-toxic amino group. Representatives of either category have been described for a large number of microaerophiles/ anaerobes, including Giardia. Reduction of the nitro group of MTZ has been extensively studied in the last 40 years (reviewed by Leitsch, 2019) and several activation pathways common to the majority of microaerophiles/anaerobes have been described in detail (see Section 2.1). Most prominently involved is the PFOR– ferredoxin couple which exists in virtually all microaerophiles/anaerobes (Narikawa, 1986), including Giardia. PFOR decarboxylates pyruvate to acetyl-CoA, and concomitantly, transfers electrons to ferredoxin, an electron shuttle protein. Ferredoxin, in turn, has a very low redox potential and can transfer electrons to the nitro group of MTZ, thereby activating it. This drug activation pathway was shown to function also in Giardia (Townson et al., 1994, 1996). In accordance with this finding, a specific knock-down of PFOR expression in Giardia by using hammerhead ribozymes (Dan et al., 2000) has been shown to reduce sensitivity to MTZ. This effect, however, was accompanied by a major shift in cellular physiology as indicated by a surprisingly high oxygen tolerance in PFOR knock-down cells. This makes it difficult to distinguish between direct and indirect effects of the PFOR knock-down on MTZ susceptibility. TrxR, another nitro drug activating enzyme in microaerophilic parasites (Leitsch et al., 2007, 2009), including Giardia (Leitsch et al., 2011; Section 2.1) has also been shown to reduce 5-nitroimidazoles, including MTZ, furazolidone (Leitsch et al., 2016) but not nitazoxanide (Leitsch et al., 2016). Accordingly, overexpression of TrxR can render Giardia significantly more sensitive to MTZ and furazolidone, but not to nitazoxanide (Leitsch et al., 2016). Finally, NR-1 which has an FMN-binding domain and a ferredoxin-like domain with a predicted 4Fed4S iron-sulphur cluster can render Giardia and E. coli more sensitive to MTZ (Nillius et al., 2011) and at least E. coli more sensitive to nitazoxanide (Muller et al., 2015; Nillius et al., 2011), although the extent observed can vary between experiments (Muller and Muller, 2019). Another, just recently

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described nitroreductase which lacks the ferredoxin-like domain of NR-1 (Muller and Muller, 2019), nitroreductase 3 (NR-3), has also been shown to render E. coli more susceptible to MTZ. Surprisingly, neither recombinant NR-1 nor NR-3 can reduce MTZ in vitro although at least the former displays marked nitroreductase activity with dinitrotoluene (Muller et al., 2007a). It also does not reduce nitazoxanide but is bound (Muller et al., 2007b) and strongly inhibited by the drug. The inability of recombinant NR-1 to reduce MTZ might, however, might be caused by inadequate or insufficient synthesis of its iron-sulphur cluster in E. coli. A nitroreductase with nitro drug deactivating function, nitroreductase 2 (NR-2), has also been characterized in Giardia (Muller et al., 2013, 2015). Overexpression of NR-2 in Giardia increases tolerance to MTZ approximately threefold, and its overexpression in E. coli results in total insensitivity to MTZ (Muller et al., 2013). Thus, increased expression of this enzyme can be interpreted as an active resistance mechanism. Despite having the opposite role, NR-2 is closely related to NR-1, all three NRs characterized being of bacterial origin (Muller and Muller, 2019). In analogy with NR-1, NR-2 also depends on its ferredoxin domain for activity (Muller et al., 2013). Both enzymes reduce nitro groups but NR-2 transfers a total of six electrons resulting in the formation of an amino group. This capability of NR-2 was directly demonstrated in vitro with recombinant NR-2 reducing 7-nitrocoumarin to 7-aminocoumarin (Muller et al., 2015). Quite puzzlingly, NR-1 and NR-2 seem to occur in an enzyme complex (Muller et al., 2015). In terms of nitroheterocyclic drugs, no credible evidence has been presented for any further active resistance mechanism in Giardia so far. 3.1.4 Molecular characterization of resistant cell lines The four most intensely studied Giardia resistant cell lines are 106-2ID10 (Boreham et al., 1988), 713-M3 (Townson et al., 1992), WB-M3 (Townson et al., 1992), and C4 (Muller et al., 2007a). The first three cell lines were generated as MTZ-resistant cell lines whereas C4 was generated as a nitazoxanide-resistant cell line and was found to display cross-resistance with MTZ. These cell lines have proven to be highly instrumental in assessing the importance of the above presented resistance mechanisms. By subjecting lines to transcriptional (Ansell et al., 2017; Muller et al., 2008, 2013), proteomic (Emery et al., 2018; Leitsch et al., 2011; Muller et al., 2019), and biochemical analyses (Ellis et al., 1993; Leitsch et al., 2011; Muller et al., 2018) and making comparisons between drug sensitive parent and resistant cell lines, we have learned a lot about resistance

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mechanisms (Ansell et al., 2017; Emery et al., 2018). Although the resulting data from these studies have often been difficult to interpret, with different observations seen between cell lines and laboratories (Emery et al., 2018; Muller et al., 2019) (Table 1), they have highlighted the complexity of nitroheterocycle drug resistance, suggesting that resistance can be achieved via several independent routes. They have also identified differentially expressed genes, e.g., ABC transporters (Emery et al., 2018), that likely play a role in the emergence of nitroheterocycle resistance. As an example of the complexity of resistance mechanism detected to date, no single drug activating pathway, i.e., neither PFOR, nor TrxR or NR1, has been found to be implicated in all resistant cell lines and in all studies. Interestingly, NR2 has not yet been found to be associated with resistance to this drug class. While this may be associated with the very low expression of NR2, it would appear that this protein is not a mediator of resistance. Also matching the sets of differentially regulated genes in the respective susceptible and resistant cell line couples gave puzzling results (Emery et al., 2018; Muller et al., 2019). The overlap of the differentially expressed gene sets of 106:106-2ID10, 713:713-M3, and WB: WB-M3 was very small in one study, amounting to only seven genes (Emery et al., 2018). In another study (Muller et al., 2019), the differentially expressed gene sets of 106:1062ID10 and 713:713-M3 had an overlap of 16 genes when grown in the presence of MTZ and only five when grown in the presence of nitazoxanide. No overlap at all was found between the sets obtained with MTZ and nitazoxanide, respectively. Finally, the overlap of the differentially expressed gene sets between the two laboratories was also minimal, with variant surface protein 88 (VSP-88) being the only candidate. This is a disappointing outcome, considering that the number of differentially expressed genes within each strain couple was high, i.e., ranging from 76 (Emery et al., 2018) to more than 415 (Muller et al., 2019) genes. Biochemical analyses of resistant cell lines have also been performed (Ellis et al., 1993; Leitsch et al., 2011; Muller et al., 2018). While these studies have demonstrated some similarities between resistant parasites, particularly around changes in oxygen scavenging capacity, they support previous findings of resistance mechanism complexity. PFOR activity and NADPH-dependent reduction of flavins has been found to be decreased in 106-2ID10 (Ellis et al., 1993) and 713-M3 (Ellis et al., 1993; Leitsch et al., 2011) as compared to their respective parent cell lines. NADPHdependent reduction of flavins is associated with oxygen scavenging because reduced flavins quickly react with molecular oxygen to form hydrogen peroxide. This enzyme activity was found to be a factor of clinical MTZ

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resistance in T. vaginalis (Leitsch et al., 2014). However, a homologue of the T. vaginalis enzyme flavin reductase 1 (FR1) which exerts this activity is not present in the G. lamblia genome necessitating further research. The resistant cell line C4 was also found to have impaired oxygen scavenging pathways, lower FAD levels, and decreased nitroreductase activity as compared to its WB C6 parent strain (Muller et al., 2018). Further, NAD+, NADP+ and NADPH concentrations were found to be decreased in C4, indicating a major shift of the metabolism which was further indicated by a steep decrease of ornithine carbamyl transferase (OCT) activity. These findings, at least partially, mirror findings in T. vaginalis where MTZ resistance was linked to decreased intracellular FAD concentrations and defective oxygen scavenging (Leitsch et al., 2009, 2010, 2014). It is also important to note that the combination of impaired nitroreductase activity on the one hand, and decreased levels of cofactors (NADPH) and coenzymes (FAD) on the other, must result in a much slower activation of nitroheterocycles, i.e., in resistance. To conclude, in the last 10–15 years our understanding of nitroheterocycle resistance in Giardia has greatly improved. However, as the process is complex, it remains impossible to map resistance to specific genetic markers, a shortcoming which is greatly deplored in the clinic (Nabarro et al., 2015) and that hampers the development of appropriate diagnostic tools.

3.2 Benzimidazoles 3.2.1 The phenomenon of benzimidazole resistance Benzimidazoles are commonly used to treat Giardia infections. Clinical trials and experimental reports have shown that ABZ and MBZ are leading agents of this class; however, others including oxibendazole, oxfendazole, fenbendazole, nocodazole, flubendazole and parbendazole also exhibit strong anti-Giardia activity. Non-carbamate benzimidazoles which include thiabendazole and triclabendazole have a lower activity. Benzimidazoles, particularly ABZ and MBZ, have less severe adverse effects than nitroheterocyclic agents such as MTZ being hence better tolerated (Karabay et al., 2004; Misra et al., 1995). Further these drugs show higher potency in vitro, i.e., minimal lethal concentrations (MLCs) in the sub-μM range, and have lower curative doses in experimental giardiasis (Reynoldson et al., 1991). Thus, after their initial introduction as antihelmintics in the 1970s then as anti-Giardia agents in the late 1980s, ABZ and MBZ have been widely used anti-parasitic agents. Indeed, they

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have seen widespread use at single dose (400–600 mg) from almost four decades in mass drug administration programs (MDA) in Africa, Asia and Latin America for control of soil transmitted helminths (STHs: Ascaris, Ancylostoma/Necator, Trichuris). However their efficiency has declined over the period 1995–2015 (Schulz et al., 2018). As with nitroheterocyclic anti-Giardia drugs therapeutic failures with benzimidazoles have been reported but to a much lesser extent (Brasseur and Favennec, 1995; Kollaritsch et al., 1993) and are considered multifactorial. Resistance to these compounds is associated with host factors (including compliance, immunosuppression, intestinal microbiota dysbiosis and drug metabolism) and the parasite factors (such as reinfections, variability in drug susceptibility and true resistance) (Arguello-Garcia et al., 2004). Further, inadequate use of these drugs is also likely to play an important role in treatment failures and that resultant emergence and spread of resistant Giardia parasites, particularly in emergent economy countries where the prevalence of giardiasis and STHs reaches >20%. For example, in Mexico and Peru, ABZ or MBZ are still given at suboptimal (single) doses in deworming MDA strategies without monitoring anti-Giardia effectiveness (Bailey et al., 2013; Flisser et al., 2008). Also, in Bangladesh the administration of MBZ at single dose (600 mg; two monthly-intervals during 1 year) in children may account for the increase in the prevalence of giardiasis from 4% to 49% (Northrop-Clewes et al., 2001). Further, there are 8–46% commercial formulations of ABZ, MBZ and nitazoxanide of low quality (i.e. with lower content of drug) that are used nationwide in Ethiopia (Suleman et al., 2014) and therapeutic failures with ABZ have been reported (18 out of 20 cases) in people who have returned from Asia, Africa and Central America to Austria (Kollaritsch et al., 1993). Collectively, these data recognize that benzimidazole resistance in Giardia is an important issue with a magnitude and impact which has not yet been truly defined. In addition to monotherapy it is also important to recognize that ABZ has been used in combination with other drugs to treat patients with nitroimidazole-refractory giardiasis and that parasitological cure does not always occur. In an outbreak in Norway, 30 out of 38 patients who did not respond to MTZ were cured with a MTZ-ABZ combination (Morch et al., 2008). In addition, in a recent study in children with giardiasis who were unsuccessfully treated with MTZ, tinidazole or secnidazole, and as a result treated with a combination of secnidazole-ABZ demonstrated a cure 82% (resolved infection in 9 out of 11 cases) (Escobedo et al., 2018). Thus, the

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existence of ABZ- and multidrug-resistant parasites are issues that have to be considered in monotherapy and combination therapies. Detailed biochemical and molecular studies of isolates from benzimidazole-refractory giardiasis cases have not been reported. The only study so far reported includes the characterization of an isolate obtained from a patient with giardiasis who had acquired immunodeficiency syndrome and that was subjected to multiple therapies with MTZ (7), secnidazole (1), MTZ-ABZ (1) and nitazoxanide (2). This parasite isolate displayed a 1.35–1.90 higher in vitro IC50 value for ABZ in a CaCo-2 co-culture model and a 4.55 fold higher inhibitory ABZ dose (ID50) in a neonatal mice model as compared to clinically ABZ-susceptible isolates (Abboud et al., 2001). In spite of the phenotypic in vivo-to-in vitro reflection of ABZ resistance observed in this study, ABZ-resistant Giardia isolates with a resistance index of >10 were not established. However, this may be the result of a resistance fitness cost that impaired the ability of highly resistant trophozoites to grow in these settings (Tejman-Yarden et al., 2011). For this reason all ABZ resistance characterization studies have been performed using isogenic cultures developed in the laboratory. 3.2.2 Benzimidazole resistance induced in the laboratory All axenic ABZ resistant parasite lines have been generated in the laboratory by exposing trophozoites to sub-lethal concentrations of ABZ. In 1996, Lindquist selected for ABZ resistance by exposing the WB strain to ABZ concentrations of up to 4.5 and 9 μM (37.5 and 75 times wild-type IC50). Parasites were exposed to drug using a one-day intermittent exposure cycle and increasing the concentration of ABZ until the maximum values were reached. This resistance was retained when the trophozoites were grown in drug-free medium for <10 weeks (4.5 μM) and 1 week (9 μM) (Lindquist, 1996). In other studies carried out by Upcroft and colleagues a constant exposure to increasing concentrations of ABZ was used to generate two ABZ-resistant lines from Australian isolates (Upcroft et al., 1996b). These isolates, BRIS/83/HEPU/106-Alb and BRIS/91/HEPU/1411-Alb grew under constant ABZ exposure (0.8 μM ABZ). Another ABZ resistance strain derived from MTZ resistant WB (WB1B-M3-Alb) was also induced. This strain grew in the presence of 2 μM ABZ and after >8 months, was shown to be cross-resistant to parbendazole (Upcroft et al., 1996b). The strategy of continuous trophozoite subculture under increasing ABZ pressure has also been used to obtain a series of ABZ-resistant clones,

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WBRA1.35 (resistance index 4.1), WBRA8 (resistance index 24.7) and WBRA250 (resistance index 771.6) that were obtained after 160, 322 and 534 weeks of adaptation, respectively (Arguello-Garcia et al., 2009; Paz-Maldonado et al., 2013). While these clones retained ABZ resistance for at >3 months in ABZ-free medium, other studies, have suggested that drug pressure is required for the maintenance of ABZ drug resistance. A series of ABZ-resistant lines derived from Portland-1 strain (P-1R) and 8 Mexican isolates (IMSS-1R to IMSS-R4 and INP-R1 to INP-R4) for example (IC50 values of 4.52–5.58 times higher that their sensitive wild-type parents), were shown to revert to a sensitive phenotype after 8 weeks in drug-free medium ( Jimenez-Cardoso et al., 2009). Genomic data (GiardiaDB; Morrison et al., 2007), transcriptome analyses and additional “omics” methodologies including quantitative proteomics and representational difference analyses of gene expression (RDA) have been used to investigated the ABZ resistant mechanisms in in vitro derived ABZ-resistant parasites. While these studies may be criticized given assemblage A1 parasites are less variable (Saghaug et al., 2019) and seen less frequently in humans than other infecting G. duodenalis assemblages (Caccio and Ryan, 2008); they have provided some basis for resistance mechanisms that this parasite might evolve in the field. 3.2.3 Factors of benzimidazole resistance For benzimidazoles, it is suitable to classify resistance mechanisms as pharmacokinetic (PK) or pharmacodynamic (PD) considering that the preferential targets of these drugs are known. The first type includes drug-related processes such as altered uptake/efflux, lower activation or higher inactivation of the drug or its active metabolites. The second type involves target-related processes as sequence/structure changes, altered synthesis rate or activity of molecules implicated in its downstream function. This classification also provides a platform for holistic drug evaluations in therapeutics (Asin-Prieto et al., 2015). The mode of action of the benzimidazole carbamates against Giardia, helminths and fungi is through an interaction with microtubule elements at a site in β-tubulin monomer involving three conserved residues (Phe167, Glu198 and Phe200). This interaction inhibits microtubule dynamics and as a result impairs multiple essential functions including motility, cytokinesis and molecular trafficking (Diawara et al., 2013; Lubega and Prichard, 1990; MacDonald et al., 2004; Robinson et al., 2004). Interestingly ABZresistant lines derived from WB do not harbour mutations at Phe200 (WB1B-M3-Alb; Upcroft et al., 1996b) nor any other ABZ-tubulin

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interacting position (WBRA250; Arguello-Garcia et al., 2009). However, sequence changes were identified in β-giardin, an annexin-like family of microtubule-associated proteins present in Giardia (Peattie, 1990) in a ABZ resistant P-1R line ( Jimenez-Cardoso et al., 2009). The C-terminal or ROD domain of β-giardin harboured most of the mutations with the sequence IDRPRE being permanently changed to TIARER; however these changes did not revert when the P-1R line was removed from drug pressure with a corresponding increase in sensitivity ( Jimenez-Cardoso et al., 2009). While this observation suggests that mutations in β-giardin might not be responsible for ABZ resistance, further studies are required to ascertain this observation. Interestingly, benzimidazole-resistant Vittaforma corneae parasites have also been shown to harbour a Glu198Gln mutation in β-tubulin (Franzen and Salzberger, 2008). In a broader sense, cytoskeletal changes are likely to play a significant role in ABZ resistance given WB1BM3-Alb trophozoites display a distinct pattern of α-tubulin distribution, including a more prominent median body than ABZ sensitive parasites (Upcroft et al., 1996b). Thus, it is still feasible that a non-genomic but epigenetic change, causes alterations in microtubule architecture or dynamics, e.g., post-translational modifications (PTMs) as poly-glycylation or acetylation, playing thus a role in ABZ resistance in this parasite. As the activity of ABZ is dependent on its biotransformation (dioxygenation) into the pharmacologically active ABZSO and the inactive ABZ-sulphone (ABZSOO) metabolites, it is possible that parasites resistant to ABZ have a mechanism to reduce ABZ biotransformation. In higher eukaryotes ABZSO is formed as R(+) and S() enantiomers by the action of flavin monooxygenases (FMO) or cytochrome P450 oxygenases (CYP450). In humans this includes CYP3A4 and CYP1A1 and in rats CYP2C6 and CYP2A1. Further, CYP450 are involved in complete type-1 detoxification reactions by converting ABZSO into ABZSOO (Capece et al., 2009; Fargetton et al., 1986; Rawden et al., 2000). While sequence homologues of FMO or any CYP450 isoform have yet to be detected in GiardiaDB, other enzymes in this parasite may be able to metabolize ABZ and hence may be associated with resistance. Evidence that such enzymes exist in Giardia include: (a) immunoelectron microscopy studies which have shown a differential distribution of parent compound and metabolites (Oxberry et al., 2000), and (b) intracellular concentrations of ABZ and metabolites in lysates of WB-DMF and WBRA250 clones which have shown a similar uptake of ABZ but differential production/ accumulation of ABZSO and ABZSOO (Arguello-Garcia et al., 2015).

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These observations indicate that an enzyme(s) containing either a FAD (FMO-like) or heme (CYP-like) prosthetic groups are involved in ABZ “activation” into ABZSO and “deactivation” into ABZSOO and that the expression/activity of these molecule(s) is lower in ABZ-resistant trophozoites. Another critical factor in Giardia parasite ABZ resistance is the ability of the parasite to deal with oxidant stress. In addition to targeting microtubule elements, ABZ and MBZ are believed to induce oxidative stress in host liver (Locatelli et al., 2004) and in helminths. To protect themselves from this damage cell increase the activity of antioxidant enzymes including peroxidase, glutathione peroxidase, catalase and superoxide dismutase (Bartikova et al., 2010). While these “conventional” antioxidant enzymes are not found in Giardia; an alternative repertoire of enzymes including superoxide reductase (gSOR), two 2-Cys-Peroxiredoxins (gPxrds), 1a and 1b, and several lateral transfer candidates (LTC) including a flavohemoglobin, an NADH oxidase (gNADHox) and an A-type flavodiironprotein (gFDP) as well as cysteine, as low-molecular weight thiol (Mastronicola et al., 2016) have been identified in parasites. There is also clear evidence that ABZ promotes oxidative stress in Giardia by mediating the production of reactive oxygen species (ROS) that induce oxidation of parasite’s proteins and cause nucleic acid damage. Treatment of parasites with ABZ results in histone H2AX phosphorylation, 8-deoxyguanosine adduct formation and extensive DNA fragmentation (Martinez-Espinosa et al., 2015). In addition, there is evidence that ROS are depleted in ABZ-resistant lines including WBRA250 (Arguello-Garcia et al., 2015). Quantitative proteomics and end-point PCR have also shown an overexpression and up-regulation of PXrd1a, NADHox and FDP in these parasites (Arguello-Garcia et al., 2015). Based on the ROS specificity of these latter enzymes, it appears that molecular oxygen (O2); converted into H2O by NADHox and gFDP and hydrogen peroxide (H2O2; scavenged by gPxrd1a) are the main ROS elicited by ABZ in trophozoites and that ABZ-resistant parasites are able to avoid the deleterious effects of these ROS. Superoxide anion (O–2; processed by gSOR) does not appear to participate in this process. The biological implications of the overexpression of O2 and H2O2scavenging enzymes to evade oxidative damage in ABZ-resistant parasites is likely related to the impact of these enzymes on the production of hydroxyl radicals (OH.-), which is considered the most cytotoxic ROS (Ansell et al., 2015; Edwards, 1993). As the conversion of O2 and H2O2 into OH can be inhibited by NADHox and FDP, increased activity of these enzymes restricts the production of O 2 and H2O2. As H2O2 is not produced SOR activity is

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also not induced (Fig. 1A). If H2O2 is produced, it can be scavenged by high levels of Pxrd1a. In this way parasites prevent the interaction of H2O2 with divalent cations such as Fe+2 or Cu+2 that results in the formation of OH which in turn reacts with and oxidizes DNA and proteins. The increased pool of thiols (including cytosolic free cysteine) observed in ABZ-resistant trophozoites also provides additional ROS scavenger capacity.

A

Rb– Flavodiiron Fe protein Rb

dO2

FAD

dO2

NAD(P)

FAD

H2O

dO2 DT diaphorase

NAD(P)

O2•–

H2O

NADH oxidase

NAD(P)H

NAD(P)H

B

H2O

H2O2

SOR Rb–

Peroxiredoxin 1a

Fe

FAD

Rb

Trx-SH Trx-SS Fe2+ Fe3+

–

OH

H2AX phosphorylation 8OHdG adducts

Protein carbonylation

Fig. 1 Molecules involved in G. duodenalis ABZ-resistant clones. (A) Partial scheme of the antioxidant system in G. duodenalis trophozoites. Three H2O-forming enzymes (indicated within green rectangles) have been observed to be up-regulated at the mRNA level. The increase protein levels of these molecules in ABZ-resistant clones point to a higher capacity to scavenge dissolved Oxygen (dO2) and Hydrogen peroxide (H2O2) to avoid formation of hydroxyl radicals ( OH) which in turn may cause DNA damage and protein misfolding through carbonyl formation. (B) Up: Protein model of gARR-VSP (Vsp 116). The transmembrane domain (aa 658–694, displayed in orange), cytoplasmic tail CRGKA (aa 695–699, magenta), EGF-like domain signature-2 (aa 302–317, cyano) and cytocrome P450 cysteine heme-iron ligand signature (aa176–185, green) are indicated. Bottom: Magnified view of a favoured docking position (ΔG ¼  5.53 kCal/mol) of hemin (protoporphyrin) with the heme-iron ligand moiety of gARR-VSP. Abbreviations: Rb ¼ rubredoxin, H2AX¼ histone 2A, 8OHdG ¼ 8-deoxyguanosine, SOR ¼ superoxide reductase, Trx ¼ thioredoxin.

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3.2.4 Molecular characterization of benzimidazole-resistant cell lines Three ABZ-resistant parasite lines have been partially characterized at the biochemical and molecular level. Karyotype-Southern blot analysis of the double-resistant WB1B-M13-Alb line has demonstrated that this parasite has a duplication and rearrangement of a chromosome 6-specific sequence (Upcroft et al., 1996b). Whereas the examination of the Mexican isolate INP-1R identified structural changes in β-giardin ( Jimenez-Cardoso et al., 2009). ABZ-resistant clones derived from WB strain that do not harbour mutations in β-tubulin exist and transcriptional studies have identified the differential expression of GL50803_101765 (annotated as Vsp116), named “ABZ resistance related VSP (ARR-VSP)” (ArguelloGarcia et al., 2009) when comparing ABZ-resistant and sensitive parasite lines. Interestingly, GL50803_101765 encodes a protein with an epidermal growth factor type 2 (EGF-2) and a CYP450-type cysteine heme-iron ligand signature (Fig. 1B). Based on this observation, ARR-VSP may be a surface-exposed protein that participates in heme capture/acquisition similar to LTC flavohemoprotein and cytochrome B5 isoforms (Rafferty and Dayer, 2015). Further studies with these ABZ-resistant clones identified additional up-regulated proteins associated with the structure and regulation of cytoskeletal functions including α2-giardin, a member of the calcium-binding group E annexins that is located at plasma membrane and flagella of trophozoites (Weiland et al., 2005); and GTPase Ranbinding protein 1, a small GTPase that is known to regulate mitotic processes as spindle assembly in eukaryotes (Zhang et al., 2014). Other transcripts such as the one encoding triosephosphate isomerase (TPI), the rate-limiting enzyme for glycolysis, were drastically down-regulated in resistant clones (Paz-Maldonado et al., 2013). By comparative proteomics of total cell lysates, protein expression patterns of the mentioned molecules paralleled that of its corresponding transcripts (Paz-Maldonado et al., 2013) indicating that transcriptional and translational processes are coupled in ABZ-resistant trophozoites. In summary, although some key mechanisms surrounding benzimidazole resistance in G. duodenalis are unravelling, questions still remain. While we are beginning to understand the impact of ABZ and its metabolites on the parasite cytoskeleton (Crossley et al., 1986; Oxberry et al., 2000) the specific role of several molecules and their underlying processes (e.g. drug transporters/pumps, epigenetics and signal transduction pathways) have yet to be clarified. In addition, it will be important to include in future studies clinical isolates obtained from benzimidazole-refractory patients.

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4. Drugs under investigation for repurposing in giardiasis The majority of the currently used anti-Giardia drugs have been repurposed. That is they became anti-Giardia compounds as a result of previously described activity for a different indication. These drugs include MTZ, furazolidone and nitazoxanide that were first used against anaerobic bacteria; quinacrine that was formerly an antimalarial agent; paromomycin which was first used as an antimicrobial agent and ABZ/MBZ which were originally used as anthelmintics. The success of drug repurposing in identifying new anti-Giardia compounds has meant this it has remained a mainstay mechanism for the identification of new novel drugs to treat giardiasis. Given concerns around the declining efficacy of anti-Giardia drugs, a number of research groups have instigated studies to search for new agents against G. duodenalis from clinically approved drugs. Drug repurposing has many advantages over the investigation of novel chemical entities including reduced development costs (reviewed in (Andrews et al., 2014). Drug repurposing drug discovery also opens interesting perspectives such as a ‘collateral benefit’, i.e., controlling (psycho)-somatic illnesses with concomitant parasitological cure, however shared molecular targets in the host and parasite may not always be of benefit. Of relevance to anti-parasitic drug discovering which often identifies general antimicrobial agents as candidates (see above), are potential collateral effects on host microbiota which may be a liability. An additional difficulty may also lay in the unique protein landscape (half of the predicted proteins expressed by Giardia parasites have no predicted function/annotation) of Giardia parasites as compared to other organisms. In this context, from a current repertoire of 2538 small molecule drugs clinically approved and included in DrugBank database (https://www. drugbank.ca›stats), only 66 have been predicted to be directed against 15 GiardiaDB targets (Sateriale et al., 2014). Despite this limited landscape, a series of FDA-approved drugs have been tested for antigiardial activity and some have demonstrated promising potential as anti-Giardia agents.

4.1 Anti-obesity agents Orlistat or tetrahydrolipstatin (trade names: Xenical®, Alli®) is the saturated derivative of lipstatin, a potent natural inhibitor of gastric and pancreatic lipases that break triglycerides into absorbable fatty acids in the intestine. It is used to treat overweight and obese humans in combination with a

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reduced-calorie diet. Orlistat is isolated from the Gram-positive bacterium Streptomyces toxytricini (Barbier and Schneider, 1987), and it has good safety due in part to its poor intestinal absorption. Interestingly orlistat has also been shown to inhibit the growth of apicomplexan and trypanosomatid parasites (Miculka et al., 2011). It has also recently been demonstrated to inhibit the growth of standard (WBA6, assemblage A1; in vitro IC50 2.8 μM) and metronidazole resistant (14–03/F7, assemblage A2, IC50 6.2 μM) Giardia parasites at lower concentrations than that of MTZ (4.3 and 11.0 μM, respectively) (Han et al., 2013). The rationale for the anti-giardial activity of orlistat is based on the likely inhibition of enzymes related to lipid metabolism. While Giardia parasites have a limited complement of enzymes for lipid uptake, synthesis and conversion (Yichoy et al., 2011), there are five (assemblages A2 and B) to seven (assemblages A1 and E) lipases in GiardiaDB that could be targeted by orlistat. Investigations into the anti-Giardia activity of orlistat have also demonstrated that this drug causes blebbing at dorsal surface and flagella tips of trophozoites, observations that are consistent with its proposed sequelae on membrane lipid integrity (Hahn et al., 2013). Importantly, giardicidal concentrations of orlistat (>14 μM for WBA6 and >43 μM for 14–03/F7) can be reached at the gut lumen under accepted anti-obesity regimens (Hahn et al., 2013). This may suggest that individuals receiving orlistat for obesity may be protected against Giardia, however, at present, there is a little information regarding the relationship between giardiasis, human obesity and orlistat use (Muhsen and Levine, 2012).

4.2 Anti-rheumatic agents Auranofin is an organic gold compound which was developed in 1975 for the treatment of rheumatoid arthritis (Finkelstein et al., 1976). It was approved in 1982 and is available under the brand name Ridaura®. Auranofin is taken orally in doses of 6 mg daily. Auranofin became interesting for wider use when it was discovered to be a highly effective inhibitor of human TrxR (Gromer et al., 1998) and of thioredoxin-glutathione reductase in parasitic worms (Alger and Williams, 2002; Rendon et al., 2004). Auranofin was also found to kill schistosomes in vitro and to reduce schistosome loads in mice (Kuntz et al., 2007). In 2013 auranofin was also found to be active against E. histolytica (Debnath et al., 2012) and Giardia (TejmanYarden et al., 2013) in high-throughput drug screens with a drug library from Iconix Biosciences. Importantly, auranofin was shown to be effective

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against MTZ-resistant Giardia (Tejman-Yarden et al., 2013). In congruence with earlier observations, auranofin was also shown to inhibit parasite TrxR (Debnath et al., 2012; Tejman-Yarden et al., 2013). A recent phase I trial designed to evaluate auranofin’s applicability as a short-term treatment for giardiasis and amoebiasis has shown that this drug is safe when administered in doses of 6 mg/d for 7 days (Capparelli et al., 2017). Concentrations of auranofin in plasma and stool samples were also shown to increase steadily throughout treatment. Thus, 7-day treatment courses with auranofin are a realistic prospect, rendering auranofin an excellent candidate for antigiardial and antiamoebal therapy in the future. Although auranofin has been shown to be a potent inhibitor of Giardia TrxR activity in enzyme assays the mode of action of this drug in microaerophilic parasites is still not completely understood. It is uncertain, for example, whether this drug inhibits Giardia TrxR in the living parasites. Considerable TrxR activity has been detected in parasites exposed to high doses of auranofin for several hours (Leitsch et al., 2016). Moreover, 10-fold overexpression of TrxR in Giardia has been shown to have no impact on the sensitivity of parasites to auranofin (Leitsch et al., 2016). In contrast, the activity of auranofin on Giardia and T. vaginalis was found to be strongly and inversely correlated to the level of cysteine in the growth medium, indicating that auranofin can indiscriminately react with sulfhydryl groups, either in free thiols or in proteins (Leitsch, 2017). Thus, further research is necessary to reveal auranofin’s mode of action against Giardia and other microaerophilic parasites. Finally, it is important to note that resistance to auranofin in microaerophilic parasites has yet been reported.

4.3 Anticancer agents 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), a derivative of 7-nitro-2,1,3-benzoxadiazole (NBD) and a potent inhibitor of human glutathione-S-transferases (Ricci et al., 2005) is a highly effective anti-cancer agent (Turella et al., 2005) which has also demonstrated activity against Giardia (Lalle et al., 2015). While the mode of action of NBDHEX against Giardia parasites remains under investigation current data suggests that it inhibits glycerol-3-phosphate dehydrogenase (Lalle et al., 2015). It is also appears to be reduced, in a similar fashion to the 5-nitroimidazoles, by Giardia TrxR after which it binds covalently to a number of proteins (Camerini et al., 2017) via one or two cysteine residues. The set of proteins bound by NBDHEX includes TrxR and elongation factor 1-γ (EF1-γ), both

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of which have been previously identified as targets of MTZ and other 5-nitroimidazoles in Giardia (Leitsch et al., 2012). Ornithine carbamoyl transferase, α-tubulin, arginine deiminase, PFOR, and axoneme-associated protein GASP-180 have also been shown to bind to NBDHEX. Further, NBDHEX has been shown to inhibit the disulphide reductase activity of TrxR, giving rise to thioredoxin reductase an activator (through the nitroreductase activity of thioredoxin reductase) as well as a drug target of NBDHEX (as a disulphide reductase). Strikingly, all these findings closely mirror what has been observed earlier with MTZ and other 5-nitroimidazoles (Leitsch et al., 2007, 2009, 2011), suggesting a similar mode of action. It is currently unknown, however, if NBDHEX can overcome MTZ resistance in Giardia. Future studies are needed to demonstrate this and to determine if NBDHEX can inhibit Giardia in vivo.

4.4 Proton pump inhibitors In recent years, several drugs that are known to act in the gut have gained attention as anti-parasitic candidates. Among these are proton pump inhibitors (PPI) that irreversibly block the H+/K+ ATPases of gastric parietal cells. As these pumps are important mediators of H+ secretion in gastric lumen, their inhibition serves to treat common gastric disorders including gastroesophageal reflux disease, peptic ulcer disease and Zollinger–Ellison syndrome (Sachs et al., 2006). Representative PPIs include omeprazole (trade names: Prilosec®, Losec®), esomeprazole, pantoprazole, lansoprazole and rabeprazole. The anti-parasitic potential of PPIs became of interested when drug susceptibility testing in anaerobic/microaerophilic protozoans including Giardia, Trichomonas, Entamoeba and Plasmodium demonstrated IC50 values in the sub-μM range (Perez-Villanueva et al., 2011; Skinner-Adams et al., 1997). In Giardia, PPIs were found to inhibit the rate-limiting enzyme of glycolysis, TPI, in a species-specific manner (Reyes-Vivas et al., 2014). Recombinant protein activity assays and site-directed mutation assessments demonstrated that omeprazole interacts with allosteric Cys222 in Giardia TPI (in human TPI it is Cys218) causing catalytic inhibition concomitant with the cytotoxic effect of this drug. The interaction of PPIs with TRI Cys222 was also shown to alter the thermal stability of enzyme rendering it more susceptible to proteolysis (Garcia-Torres et al., 2016). Importantly, a WB strain resistant to MTZ and N1-INP, an isolate resistant to nitazoxanide, displayed the same susceptibility to omeprazole as reference

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WB parasites (Reyes-Vivas et al., 2014). Further, it was shown that the protonated (activated) form of omeprazole displayed higher potency than omeprazole itself and that rabeprazole was 10 times more potent against giardial wild-type TPI than the other PPIs including omeprazole. In a structure-based drug design (SBDD) study using the PPI structure scaffold, three derivatives were synthesized in which the 5-methoxy group of the benzimidazole core was replaced by a methyl group. Substituents at the pyrimidine ring moiety were left unsubstituted (named as BHO1) or substituted as in omeprazole (BHO2) or lansoprazole (BHO3). Derivatives BHO2 and BHO3 demonstrated enhance anti-Giardia activity (IC50 around 120 μM) as compared to omeprazole (IC50 ¼ 300 μM) and formed adducts with TPI Cys22 through a covalent CdS bond involving the benzimidazole core, with or without the pyrimidine ring (Hernandez-Ochoa et al., 2017). Consistent with a mode of action against Giardia TPI, recent studies have shown that exposure of trophozoites to lethal omeprazole concentrations (>250 μM) led to higher glycogen deposition and formation of advanced glycation end products. At the ultrastructural level the main alterations were profuse cytoplasmic vacuolization and the appearance of lamellar structures suggestive of autophagy but cytoskeletal structures were non-affected (Lopez-Velazquez et al., 2019). The future evaluation of PPIs in experimental models of giardiasis is favoured by their proven tolerability and low toxicity against human cells (Hernandez-Ochoa et al., 2017). However, the use of these agents as a repurposed drug for giardiasis, should consider current knowledge and studies demonstrating that prolonged use of PPIs can lead to gastrointestinal alterations (Farrell et al., 2017). In this scenario the clinical use of PPIs in giardiasis, if approved, may require a standardized and medically controlled posology.

4.5 Inhibitors of ethanol metabolism Inhibitors of ethanol metabolism were identified as inhibitors of Giardia growth via a screen looking for compounds with activity against giardial VSP family proteins (230 members). As VSP proteins contain one or more Zn-finger motifs that have LIM- and RING-like domains and are unique surface-residing proteins of Giardia (Gargantini et al., 2016; Nash, 2002), a series of 34 Zn-finger active compounds (22 thiurams and 12 non-thiurams) were assessed for activity against parasites. Disulfiram (tetraethylthiuram disulphide; trade name: Antabuse®), with a low in vitro MLC concentration of 1.23 μM, was identified as the most active agent

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in this screen (Nash and Rice, 1998). The anti-Giardia activity of disulfiram has been confirmed by additional screens (LOPAC1280 and NIH drug libraries (Galkin et al., 2014)) and by follow-up activity studies which have demonstrated sub-μM activity (IC50 0.9 μM) with low mammalian cell (HepG2 IC50 > 40 μM) (Galkin et al., 2014). Disulfiram has also been demonstrated to be more 2–4 times active against MTZ-resistant (713 M3) than MTZsensitive (WB and GS/B) parasites (Galkin et al., 2014). It has also demonstrated in vivo activity in Giardia-infected mice, reducing trophozoite loads by 55% and 40% when administered at a doses of 5 mg/day for 4 days and 25 mg/day for 4 days, respectively (Nash and Rice, 1998). Disulfiram is an inhibitor of acetaldehyde dehydrogenase, the second enzyme after alcohol dehydrogenase in ethanol metabolism and a second-line agent for the treatment of alcohol dependence (Kitson, 1983). While disulfiram is generally well-tolerated, its combination with alcohol results in a rise of acetaldehyde that can cause a plethora of unpleasant, mid-to-severe reactions (Wright and Moore, 1990), not unlike those that have been observed when MTZ is combined with ethanol (Williams and Woodcock, 2000). The anti-Giardia mode of action of disulfiram is currently unknown. While there is at least one gene sequence annotated as acetylating alcohol/acetaldehyde dehydrogenase in GiardiaDB that may be a potential target of disulfiram, biochemical and molecular evidence suggest that, as a result of an ability to interact with cysteine residues, this drug is likely to have multiple targets in Giardia. As an example, disulfiram has been shown to interact with carbamate kinase, the third and last enzyme in the arginine dihydrolase pathway (Galkin et al., 2014). The crystal structure of carbamate kinase soaked with disulfiram has been solved (PDB ID: 4OLC), demonstrating that Cys242, located at the edge of the active site, is covalently thiocarbamoylated by disulfiram causing irreversible inhibition (IC50 ¼ 0.58 μM) (Galkin et al., 2014). Giardia TPI, is also inhibited by disulfiram (IC50  2 μM) in a species-specific manner (Castillo-Villanueva et al., 2017). Taken together current data demonstrate that disulfiram is a potent, multi-target anti-Giardia agent and a good candidate for pre-clinic trials. Further, efforts to investigate and optimize the activity of this drug for use as a treatment for giardiasis are encouraged and needed to further this work. Particular emphasis should be placed on improving the intestinal bioavailability of this drug by decreasing intestinal absorption given that this is likely to help to counteract or minimize gastrointestinal, hepatic and neurologic complications that may be caused at the usual prescribed doses (500 mg daily) (Boukriche et al., 2000).

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4.6 Beta-blockers Beta-blockers antagonize the heterotrimeric G-protein β1-adrenergic receptors of the heart by competing with their natural sympathomimetic ligands (isoprenaline, adrenaline and noradrenaline) to decrease calcium influx, reduce heart rate and lower arterial blood pressure. DL-propranolol (trade name: Inderal®) is widely used as a beta-blocker and is prescribed for high blood pressure, irregular heart rhythm and to prevent heart attacks. D-propranolol, the dextro-enantiomer of DL-propranolol has weak betablocker action. Similar to disulfiram, propranolol was one of the first clinically approved drugs assessed for activity against Giardia on the basis of its ability to adversely impact sperm motility through membrane stabilization (Hong et al., 1981). As a result of promising in vitro activity (D-propranolol IC50 ¼ 180 μM) (Farthing et al., 1987), DL-propranolol (40 mg three times a day) was combined with MTZ (400 mg three times a day for 10 days) to successfully treat a patient with MTZ-resistant giardiasis (Popovic and Milovic, 1990). It has also reportedly been used successfully as a monotherapy against MTZ-resistant giardiasis (Popovic et al., 1991). While additional studies investigating the clinical activity action of propranolol have not been reported, the promising in vitro and in vivo activity of this agent supports further work.

5. Strategies for the discovery and development of new anti-Giardia agents As currently available anti-Giardia drugs are associated with increasing rates of treatment failures, there is a need to identify new drug candidates which are active against drug resistant parasites. Mechanisms that can be used to identify and developed novel new anti-Giardia compounds include rationally designing new compounds based on validated drugs and drug targets, and screening novel chemical libraries containing hundreds or thousands of drugs and/or newly synthesized compounds (chemotypes) for anti-Giardia activity (Chen et al., 2011; Gut et al., 2011; Muller et al., 2009). Current progress in the discovery and development of new anti-Giardia drugs via these strategies will be discussed in the next sections.

5.1 Next generation synthetic compounds 5.1.1 5-Nitroimidazole and 5-nitrothiazole derivatives Synthetic nitroheterocycles such as MTZ and nitazoxanide have proven highly effective in the treatment of giardiasis. However, improvements in

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efficacy and tolerability are always possible and it has been anticipated that novel nitroheterocycles can, at least in part, circumvent resistance. There is, indeed, good evidence for this assumption as tinidazole, a 5-nitroimidazole which is closely related to MTZ, has proven to be a frequently effective alternative in the treatment of T. vaginalis infections which are refractory to MTZ (Sobel et al., 2001). Importantly, tinidazole also has a better bioavailability than MTZ and a longer serum half-life (Wood and Monro, 1975). There are also other 5-nitroimidazole drugs available of which ornidazole, ronidazole and secnidazole are presumably the most important. Ronidazole, for example, is used in the treatment of giardiasis in dogs (Fiechter et al., 2012). Interestingly, all these 5-nitroimidazoles, with the exception of ronidazole, are structurally very similar to MTZ, only differing from the latter in their imidazole ring N1 substituent (Fig. 2). As current 5-nitroimidazole drugs are so structurally similar, there has been interest in exploring chemical modifications of these agents to improve activity, particularly against drug resistant parasites. Indeed, numerous studies in this

R1

N1 NO2

R2 C2

C5

C4

N3

CH3 OH

CI

O– N

H3C N

OH

O S O O– N

H3C

N O

Metronidazole

N

N Tinidazole

N

H3C O

N Ornidazole

O

CH3

O

O– N

H2N

N

O

O– N

N

O

Ronidazole

Fig. 2 A schematic representation of 5-nitroimidazoles. The nitro group is positioned at C5 of the imidazole ring. At N1 and C2 different ligands can be attached which have pharmacokinetic and pharmacodynamic implications. Metronidazole, tinidazole, and ornidazole all have a methyl group at C2 but varied substitutions at N1. Ronidazole is an example of a 5-nitroimidazole with a more complex substitution at C2. Next generation 5-nitroimidazoles of both types have been synthetized.

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space have now been conducted and improved nitroimidazoles have been developed. While the majority of these studies have maintained the 5-nitroimidazole backbone of these compounds, some promising 4-nitroimidazole derivatives have also been synthesized (Saadeh et al., 2009). One consortium (Upcroft et al., 1999, 2006) has systematically evaluated the potential of substituents at the C2 position of the imidazole ring and replaced the methyl group of MTZ at this position with a 43 different substituents of high structural variability (Upcroft et al., 1999, 2006). The ethanol side chain of MTZ at the N1 position was changed throughout to a methyl group. Several novel compounds proved to be significantly (up to 50 to 100-fold) more effective in vitro than MTZ, notably a γ-lactam substituted 5-nitroimidazole (compound 13 in Upcroft et al., 1999) and a compound with a doubly bromated and side chain with a phenyl group (compound 17 in Upcroft et al., 2006). Strikingly, substituents with higher hydrophobicity enhanced toxicity, possibly by facilitating cell uptake. Compound 17 (C17) also proved to be highly effective against MTZ-resistant Giardia (Dunn et al., 2010; Upcroft et al., 2006). Nevertheless, it was also possible to induce low-level resistance against C17 in Giardia. Interestingly, cell lines with reduced sensitivity to C17 were hyper-resistant to MTZ (Dunn et al., 2010) although displaying normal PFOR activity and pronounced metabolization of MTZ (Leitsch et al., 2011). The same 5-nitroimidazole scaffold was used in another study in which the C2 position of the imidazole ring was exploited for the generation of novel 5-nitroimidazoles (Valdez et al., 2009). A large selection of substituents with a distal phenyl group and a bridging interjacent ethenyl or ethanyl group were synthesized and tested. Derivatives with an unsaturated bridge proved to be much more effective than those with a saturated bridge. Several of the compounds were more effective than MTZ and displayed a similar selectivity index when tested on HeLa cells. Moreover, at least four novel derivatives proved to be superior to metronidazole in terms of eradication of infection in mice (Valdez et al., 2009). Also the generation of a 5-nitroimidazole carboxamide library through addition of a carboxamide group at C2 gave a large number of potent derivatives ( Jarrad et al., 2016) of which some also proved to be highly effective against metronidazole-resistant Giardia. In addition to C2 imidazole ring modifications, the ethanol side chain at the N1 position has been harnessed as a “molecular handle” ( Jarrad et al., 2015) for the synthesis of novel 5-nitroimidazole derivatives. Replacement of the hydroxyl group with halogens or methane sulfonate (Busatti et al., 2007) resulted in potent novel 5-nitroimidazole derivatives. All synthesized

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5-nitroimidazoles were more effective against Giardia in vitro than MTZ, and at least two were also more effective against Giardia in gerbils (Busatti et al., 2013). The hydroxyl group of MTZ was also harnessed as another avenue to generate new 5-nitroimidazole libraries, i.e., through the application of “click chemistry”. To this end the hydroxyl group is converted into an azide group which can subsequently react (click) under copper catalysis with a library of alkynes and, thereby, give rise to a large number of triazole derivatives ( Jarrad et al., 2015; Miyamoto and Eckmann, 2015). This method is a rapid and inexpensive means to generate vast and unbiased structural diversity. Two different consortia independently applied the click chemistry approach for the development of novel 5-nitroimidazoles ( Jarrad et al., 2015; Miyamoto and Eckmann, 2015) resulting in the synthesis of almost 700 novel candidate drugs. Of these, a large proportion had superior characteristics as compared to MTZ with up to 500-fold stronger anti-giardial activity (Miyamoto and Eckmann, 2015), and were also clearly more active against MTZ-resistant Giardia than MTZ. Most importantly, eight out of 16 representative compounds were efficacious at clearing Giardia infections in mice, seven being more efficacious than MTZ. The same approach was also used for the synthesis of improved drugs from other nitroheterocycle drug classes, i.e., nitrothiazoles, nitrofurans, and nitropyrroles (Kim et al., 2017) with similar success (442 novel nitroheterocylces). Again, numerous compounds were identified that displayed superior characteristics as compared to the standard drug of each class, i.e., MTZ, nitazoxanide, and furazolidone, and which were active against MTZ-resistant Giardia. However, only the novel nitrothiazolides displayed little or no cytotoxicity against HeLa cells, resulting in acceptable selectivity indices. Moreover, three of six novel nitrothiazolides tested proved to be more efficacious in a mouse model than MTZ (nitazoxanide was not tested for comparison). Taken together, the click chemistry approach gave rise to more than 1100 novel nitroheterocycles of which a large proportion, arguably the majority, are superior to the standard drugs of the respective drug classes (Kim et al., 2017; Miyamoto and Eckmann, 2015). Most of the work on novel nitroheterocycles for the treatment of giardiasis has been done on 5-nitroimidazoles due to their remarkable importance for the treatment of anaerobic infections. However, nitrothiazolides have also attracted attention. In addition to the synthesis of novel nitrothiazolides by click chemistry (Kim et al., 2017) several other approaches have been used to generate promising nitrothiazolide drug candidates. Navarrete-Va´zquez et al. explored the potential of nitazoxanide benzololgues, i.e., compounds

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in which the nitrothiazole ring is expanded to a nitrobenzothiazole, through substitutions at the distal phenyl group (Navarrete-Vazquez et al., 2011). Two compounds were prepared which were more active against Giardia than nitazoxanide and displayed an improved selectivity index. The same approach was pursued in another study in which a larger structural variability was generated by substitution of the acetylsalicylic tail of the nitazoxanide benzologue with acetamide groups (Navarrete-Vazquez et al., 2015). Again, several highly potent compounds with an excellent selectivity index were produced. The acetylsalicylic tail of nitazoxanide was also replaced with other substituents in a further study (Nava-Zuazo et al., 2014) which had a similar effect as observed with the benzologues: high effectivity against Giardia and excellent selectivity indices. To conclude, research in the last 10 years has led to the discovery of a vast pool of nitroheterocycles which have display improved characteristics as compared to the established standard drugs in the respective drug class. Very likely, the problem of MTZ resistance could be successfully tackled if one or more of these novel drugs is developed and approved for clinical use. 5.1.2 Benzimidazole derivatives Benzimidazole derivatives, particularly ABZ and MBZ are well known for their anti-Giardia potency, particularly as compared with other drugs used for routine treatment of giardiasis (i.e. lower IC50 against trophozoites in vitro and lower ED50 in experimental infections). In this context, benzimidazoles with giardicidal activities usually harbour a Hydrogen at position 1 (H1) and a methylcarbamate group at position 2 while distinct functional groups are attached to position 5 (propylthio in ABZ and phenylcarbonyl in MBZ). Some exceptions are tiabendazole and triclabendazole that do not harbour a 2-methylcarbamate group. In the last two decades several studies have examined chemical modifications to the benzimidazole scaffold, mostly at the aforementioned positions. Optimization of efficacy using this strategy is likely viable given nocodazole, a benzimidazole carbamate that bears a 2-thienylcarbonyl at position 5 (used as a microtubule-acting antineoplastic agent) displays improved activity against G. duodenalis trophozoites (Arguello-Garcia et al., 2004; Morgan et al., 1993). Libraries of benzimidazole derivatives have also emerged as a strategy to investigate structure activity relationships (SAR) that can be harnessed to improve activity and selectivity. Indeed, in the last two decades multiple small libraries of ‘second generation’ anti-giardial benzimidazoles have been

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developed and assessed for activity in biological assays. In initial studies, a library of 18 synthetic benzimidazole derivatives with modifications at positions 2, 5 and 6 were generated and assessed for anti-Giardia activity. These studies demonstrated the importance of the carbamate-like groups at position 2 for interaction with tubulin because only 3 compounds (all having a 2-methoxycarbonylamino substituent) interfered with tubulin polymerization; however interestingly, all but one exhibited sub-μM IC50 values against parasites (two of which (benzimidazole itself and a 2-methylthio derivative) were more potent than ABZ (IC50 < 0.03 μM; Valdez et al., 2002). Using benzimidazole-2-alkylthio as a pharmacophore, a series of 12 derivatives with a benzimidazole-1-methyl-2-methylthio-6-Chlorine scaffold and different 5-carboxamide variants were generated. These compounds retained sub-μ M IC50 values against parasites, with 5-pyridine variants demonstrating more potent activity than ABZ (Flores-Carrillo et al., 2017). Considering the increased potency of compounds with hydrogen acceptors (2-alkylthio) and aromatic rings (pyridine, imidazole, etc.) attached to alkyl chains, a series of 19 new benzimidazole-2-alkylthioimidazoles were screened. All compounds retained sub-μM efficacy with only two derivatives (with 1-methyl and 5-ethoxy groups) performing better than ABZ (Perez-Villanueva et al., 2013). Additional modifications have also been studied but have not resulted in compounds with improved anti-Giardia activity. For example, generating 2-carbonyl variants with 1-methyl substituents (24 benzimidazole derivatives) resulted in retention but not improvements in potency (Valdez-Padilla et al., 2009). The same effect was seen with 14 synthetic benzimidazole-1H-2-mercapto derivatives containing 4,6-dibromides or 4,6-dichlorides (Andrzejewska et al., 2004). Other structural approaches to optimize the antigiardial activity of benzimidazoles, including the introduction of halogen (fluoride)-rich moieties as a mechanism to improve solubility and adsorption have been performed with data demonstrating mixed results. A study using a library of nine benzimidazole-2-trifluoromethyl derivatives of ABZ or MBZ demonstrated no improvement in activity (IC50 values, i.e., at the μM range, for all compounds in relation to that of ABZ IC50 ¼ 0.037 μM) (Navarrete-Vazquez et al., 2001). A series of nine halogenated (F, Br and/or Cl) benzimidazole derivatives with 2-trifluoromethyl or 2-pentafluoroethyl substituents were shown to have similar potency to ABZ (Andrzejewska et al., 2002). A further seven benzimidazole-2-trifluoromethyl derivatives with simple substituents at positions 1 (H or methyl), 5 and 6 (H or Cl) and seven similar derivatives with bioisosteric substituents (-Cl, -F, -CF3, -CH) demonstrated sub-μM IC50 values similar to that of ABZ (Navarrete-Vazquez et al., 2001,

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2006). However, the assessment of nine benzimidazole-2-trifluoromethyl derivatives with more voluminous substituents at positions 5 and 6 demonstrated more promising activity, with IC50 values in the nanomolar range (Hernandez-Luis et al., 2010). Despite these structural insights and the generation of some 128 benzimidazole derivatives, of which nine have shown higher potency than ABZ (i.e. at least 3-times lower IC50 values), none have been tested beyond in vitro assays. Two groups of benzimidazole derivatives with complex structures have been tested for therapeutic potential using trophozoite-cell monolayer cultures or experimental infections with Giardia in young mice. From a library of 18 synthetic bis(oxyphenylene)benzimidazoles, namely bisbenzimidazoles, three compounds displayed IC50 values against G. duodenalis trophozoites at sub-μM range and one (compound 9) was shown to have a selectivity index of 122 in relation to rat skeletal myoblasts (Mayence et al., 2011). A set of eight thieno(2,3-d]pyrimidin-4(3H)ones substituted in position 2 of pyrimidine ring with either benzimidazole-2-yl-thioethyl or benzimidazole-2yl-1-ethyl moieties (6 and 2 compounds, respectively), cured animals at dose of 0.5 μg/mL pro die for 5 days as assessed by elimination of G. muris cyst excretion in stool samples (Mavrova et al., 2010). In a prospective comparison with 5-nitroheterocycle derivatives, there is a limited pool of second-generation benzimidazoles with proven, and enhanced, anti-giardial activity. However, further in vitro studies with these derivatives, including determining their activity against drug resistant strains are warranted to support preclinical evaluations that in turn will allow their introduction as alternative drugs for giardiasis. 5.1.3 ‘Hybrid’ compounds The treatment of parasitic diseases includes, as an emerging strategy, the use molecular hybridization synthetic routes to generate compounds that contain two or more pharmacophores. Besides providing a mechanism to develop compounds with improved pharmacokinetic and pharmacodynamic properties, this strategy facilitates the development of drug candidates with additive or synergistic activity. In Giardia, some work using ‘hybrid’ compounds has been performed with currently used drugs together with other antimicrobial agents and anti-inflammatory drugs. In this context, a small library of 13 hybrid 5-nitroheterocycles (nitazoxanide) and benzimidazoles (N-methylbenzimidazole) with distinct substituents at position 2 have been generated from their corresponding N-methyl-2-nitroanilines and evaluated against G. duodenalis, T. vaginalis and E. histolytica. Nine of these compounds contained the

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N-methylbenzimidazole core substituted at position 6 by the nitrothiazolecarboxamide moiety of nitazoxanide and four compounds contained the same hybrid structure but substituted at position 5. While synergy was not evident in each case, all hybrid molecules demonstrated promising activity against all parasites thus demonstrating their potential as antiprotozoal agents (Soria-Arteche et al., 2013). The most promising antiGiardia hybrid is CMC-20 that has an IC50 of 10 nM. Proteomic studies with CMC-20 treated trophozoites showed down-regulation of cytoskeletal proteins (α- and β-tubulins as well as α1- and β-giardins) and an axonemeassociated protein, GASP-180. An apoptotic-like cell death process with altered morphology hallmarked by ventral disc retraction and flagella tip blebbing was also observed (Matadamas-Martinez et al., 2016). The benzimidazole core structure has also been hybridized with propane- and pentane-diylbisoxy-dibenzenecarboximidamides, also known as propamidine and pentamidine, respectively, which are used to treat parasitic infections such as acanthamebiasis, trypanosomiasis, leishmaniasis and babesiosis. The mechanism of action of these compounds seems to vary among organisms although pentamidine is proposed to bind to AT-rich regions of DNA and inhibit topoisomerase II in Trypanosoma (Lemke et al., 2012). Twelve propamidine-benzimidazole hybrids that displayed anti-giardial efficacy in the μM range, similar to that of propamidine (IC50 ¼ 4.5 μM) were generated. However, one compound displayed an IC50 202 times lower than propamidine (32 nM) (Mendez-Cuesta et al., 2017). In a previous study, 10 pentamidine-benzimidazole hybrids showed activities in the μM range with exception of two compounds with IC50 < 0.5 μM that were 9.3 and 10.9 times more active than pentamidine (Torres-Gomez et al., 2008). Based on these observations, there appears to be some promise for the development of a propamidine-benzimidazole hybrid with enhanced activity against Giardia. Anti-Giardia hybrid compounds that contain two pharmacophores of distinct types have also been developed. These hybrids essentially contain nitazoxanide and an anti-inflammatory drug. As pro-inflammatory processes are intertwined with protozoal infections, these compounds provide anti-parasite and anti-disease therapies. Nitazoxanide itself is interesting in this context as it essential contains two pharmacophores bound by an amide bridge: one, the 2-amino-5-nitrothiazole it responsible for anti-protozoal activity, while the other, the acetylsalicylic half is essentially a non-steroidal anti-inflammatory drug (NSAID) Aspirin® that is used to reduce pain, fever, inflammation and prevent blood clots (revised in Buer, 2014;

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Colin-Lozano et al., 2017). In this context, it is worth noting that nitazoxanide has been shown to have anti-inflammatory activities in LPS-treated mice (Fan et al., 2019). Nitazoxanide, chimeric hybrids containing the non-steroidal antiinflammatory drugs (NSAIDs) diclofenac, ibuprofen, indomethacin, naproxen and toclofibric acid have been generated. All five nitazoxanideNSAID hybrids demonstrated sub-μM IC50 values against G. duodenalis trophozoites which were five to eight times better than nitazoxanide. Nitazoxanide-indomethacin and nitazoxanide-diclofenac were the more potent hybrids. Importantly, the toxicity of these hybrids against VERO cells was also improved (89–887 times lower), facilitating in vivo trials and the demonstration of cures in infected CD1-mice (ED50 values from 1.7 μg/kg (nitazoxanide-indomethacin hybrid) to 7.66 μg/kg (nitazoxanide-naproxen hybrid)) (Colin-Lozano et al., 2017). Collectively these studies support the notion that hybrid molecules with a benzimidazole or nitazoxanide half offer a promising landscape to restructure existing standard drugs which could potentiate and even multiply the effects of current drugs. However, it is important to note that the activity of these new hybrids against drug-resistant Giardia still needs to be determined.

5.2 Phenotypic drug screening Phenotypic drug screening, forward pharmacology, or the identification of compounds with activity against whole organisms, has become an important mechanism to identify new chemical entities with activity against parasites. Indeed, this approach has become increasing popular in many fields of antiparasitic drug discovery given the limited success of target-based drug discovery approaches over recent decades (Swinney and Anthony, 2011; Zheng et al., 2013). In the case of Giardia drug discovery research, targetbased drug discovery remains of interest to many researchers (see Section 5.3). However, phenotypic drug screening is increasing as new methods to assess the activity of large numbers of compounds against parasites are developed and shared (Table 2). Historically large phenotypic drug discovery screens have been hampered by suitable anaerobic culture conditions for screening compounds in large numbers and the reliance on manual microscopy and other labour intensive growth inhibition assessment strategies (Chen et al., 2011; Gut et al., 2011). However, new phenotypic drug discovery platforms that permit the activity of compounds to be assessed against parasites in a medium to high-throughput manner have been

Table 2 Published Anti-Giardia phenotypic compound screening campaigns. #Compounds Library screened #Hits reported

• BIOMOL (now Enzo Life

• Library of pharmacologically

BonillaSantiago et al. (2008)

1520 (480 BIOMOL; 1040 NINDS)

• 48 (36 reported as new) • Dose response not

• 5 μg/mL screen for 24 h • Imaging assay requiring

performed on all hits

parasite fixing; propidium iodide label • WB parasites • Z0 factor not reported

4,096

• 73(43 selective; 11 novel; • Five concentration screen for Chen et al.

active compounds (LOPAC1,280) • NIH Chemical Genomics Center Pharmaceutical Collection (NPC)–all approved small molecule drugs (Huang et al., 2011)

• Spectrum Collection (includes 1600 drugs, experimental molecules, and natural product compounds)

References

5 IC50 < 1 μM)

• Most potent Fumagillin IC50 < 0.01 μM

48 h (61 nM–38.3 μM)

(2011)

• Bioluminescent ATP content assay

• WB and GS parasites • Z0 factor of 0.67 • 12 (8 previously identified; • 1 μM performed for 48 h • Automated fluorescence 8 with IC50 < 1 μM) microscopy requiring cell fixing and staining • WB parasites • Z0 factor of 0.54

Gut et al. (2011)

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Sciences) ICCB (Harvard Institute of Chemical and Cell Biology) known bio-actives2 • NINDS (National Institute of Neurological Disorders and Stroke) custom collection 2–75% FDA-approved

Comments

• Iconix Biosciences, Inc. Library 910 (746 drugs (human use); 164 bioactives)

(15 previously reported) • 21 drugs; 20 bioactive

• 10 μM screen for 48 h • WB parasites • Demonstrated activity of

• Dose response not

• Z0 factor not reported

• 56 (>80% inhibition

TejmanYarden et al. (2013)

auranofin

• Malaria box

400

• 22 (>50% inhibition) • 5 μM screen at 48 h Hart et al. • 3 with sub-μM IC50 values • Automated live cell imaging (2017) • BRIS/91/HEPU/1279 parasites

• Z0 value of 0.73 • Pathogen box

400

• 13 (>95% inhibition; EC50 • 0.51–3.88 μM; 1 novel • • IC50 < 1 μM) • • 5 reference compounds •

16 μM screen for 24 h Hennessey Transgenic WBC6 parasites et al. (2018) Luciferase assay Z0 factor not reported Not all known anti-Giardia compounds in set detected as hits

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performed on all hits

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Raúl Arg€ uello-García et al.

developed and are increasingly being used to screen libraries of compounds for new compound inhibitors (Bonilla-Santiago et al., 2008; Chen et al., 2011; Gut et al., 2011). Importantly, these studies are identifying promising new biologically active compounds, some of which have progressed into clinical and pre-clinical trials for giardiasis (Kulakova et al., 2014). 5.2.1 Drug-repurposing library screens Drug repurposing is an attractive drug discovery strategy in the parasite and neglected disease fields as it leverages previous research and as a result can significantly reduce the amount of time and money needed to develop a new drug (Andrews et al., 2014; Debnath et al., 2013; Roder and Thomson, 2015). For these reasons it is not surprising that many of phenotypic screens performed against Giardia parasites, have used libraries that contain compounds approved for clinical use in humans or have significant preliminary biological data (Table 2). The libraries that have been screened for compounds with activity against Giardia parasites include the BIOMOL (now Enzo Life Sciences) ICCB (Harvard Institute of Chemical and Cell Biology) Known Bioactives Library (400 diverse biologically active compounds) (Bonilla-Santiago et al., 2008), the NINDS (National Institute of Neurological Disorders and Stroke; 75% approved drugs) Custom Collection 2 Library, the Library of Pharmacologically Active Compounds (LOPAC1,280) (Chen et al., 2011), a manually procured version of the NIH Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC (Huang et al., 2011)) (2800 clinically approved small molecules (Chen et al., 2011)), a sub-set of the Spectrum Collection (1600 drugs and bioactive compounds) (Gut et al., 2011) and a library of drugs and bioactive compounds donated by Iconix Biosciences (Foster City, CA) (TejmanYarden et al., 2013). In recent years the Medicines for Malaria Venture’s (MMV) Malaria and Pathogen Boxes have also been screened for compounds with anti-Giardia activity (Hart et al., 2017; Hennessey et al., 2018). However, unlike previously screened libraries, while the Malaria and Pathogen Boxes contain compounds with demonstrated bioactivity, very few are approved drugs (Duffy et al., 2017; Spangenberg et al., 2013). Nevertheless, all compounds within these collections were specifically chosen to facilitate drug discovery and as a result contain compounds with druglike and probe-like chemotypes (Spangenberg et al., 2013). In the past 10–12 years, a total of 9000 bioactive compounds have been assessed for activity against Giardia trophozoites in medium-large phenotypic screens of bioactive libraries (Table 2). On the whole these campaigns have identified

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41

known toxins and known anti-Giardia compounds or chemical entities with biological and chemical characteristics similar to those compounds already known to be effective against Giardia (Table 2). However, they have also been successful in identifying new actives with potentially novel modes of action (Tables 2 and 3) (Chen et al., 2011; Tejman-Yarden et al., 2013). Arguably the most pormising developments from these studies has been the identifcation of fumagillin and auranfin as new anti-Giardia agents (Table 2). Fumagillin was first identifed as a potent anti-Giardia agent in 2011 (IC50 0.01 μM; Selectivity Index >10,000) (Chen et al., 2011). Follow-up studies with this antibiotic have shown that it is a potent inhibitor of both MTZ-sensitive and MTZ-resistant parasites (sub-μM IC50 and minimum lethal concentrations (MLC); e.g., GS parasite MLC of 0.26 μM; (Kulakova et al., 2014)). In addition, fumagillin has been shown to cure adult mice of giardiasis at doses lower than those required to cure mice with MTZ (curative at 120 mg/kg as compared to MTZ which was curative at 300 mg/kg for 4 days (Kulakova et al., 2014)). While the anti-Giardia target of fumigillin is yet to be determined, the senstivity of MTZ-resistant parasites to this candidate is promising and suggests that this drug is working via a mechansim different to the 5-nitroimidazoles. Indeed, it is probable that as an inhibitor of methionine aminopeptidase 2 in humans and the eukayote protozoan Plasmodium falciparum (Chen et al., 2009; Sin et al., 1997), fumigillin kills Giardia by targetting this enzyme (Kulakova et al., 2014). This idea is supported by the fact that Giardia parasites encode a single methionine aminopeptidase 2 and that the function of this enzyme is essential to parasite survival (Kulakova et al., 2014). Importantly, the methionine aminopeptidase 2 of Giardia parasites is not known to be a target of any of currently used anti-Giardia drugs. Despite a promising novel putative target and potent efficacy data, there are concerns, however, that fumagillin may be toxic in the in vivo setting (Kulakova et al., 2014). While fumagillin is licensed for human use, it is primarily used topically and its potential for mamaliam toxicity is well-recognized (van den Heever et al., 2014). There are also concerns that the use of this drug may be limited by its poor stability (Kulakova et al., 2014). For these reasons efforts are underway to investigate the potential of fumagillin analogues with more favourable tolerability and stabilty characteristics. This includes investigating compounds with reduced intestinal absorption and those that specifically inhibit Giardia methionine aminopeptidase 2 (Kulakova et al., 2014). The disadvantage of these stratergies, however, is that drug development will then involve completely new chemical entities with limited biological data. In addition

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Table 3 Drugs and bioactive compounds with anti-Giardia activity in phenotypic compound screening campaigns.

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Giardia drug resistance and treatment alternatives

Table 3 Drugs and bioactive compounds with anti-Giardia activity in phenotypic compound screening campaigns.—cont’d

Continued

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Raúl Arg€ uello-García et al.

Table 3 Drugs and bioactive compounds with anti-Giardia activity in phenotypic compound screening campaigns.—cont’d

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Giardia drug resistance and treatment alternatives

Table 3 Drugs and bioactive compounds with anti-Giardia activity in phenotypic compound screening campaigns.—cont’d

Continued

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Raúl Arg€ uello-García et al.

Table 3 Drugs and bioactive compounds with anti-Giardia activity in phenotypic compound screening campaigns.—cont’d

a

Phenotypic screen A (Bonilla-Santiago et al., 2008), B (Chen et al., 2011), C (Gut et al., 2011), D (Tejman-Yarden et al., 2013), E (Hart et al., 2017), F (Hennessey et al., 2018).

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47

there is still no clear evidence that compounds with reduced intestinal absorption make for better anti-Giardia drugs. MTZ, our current goldstandard anti-Giardia chemotherapeutic has an oral bioavailabililty of >90% with peak plasma concentrations obtained in 2–3 h (Lee et al., 2010). Auranofin was first identified as a potential anti-giardiasis chemotherapeutic in 2013 after a library of drugs and bioactive compounds donated by Iconix Biosciences (Foster City, CA) were screened for compounds with activity against Giardia trophozoites (Tejman-Yarden et al., 2013). As auranofin was approved for the treatment of rheumatoid arthritis (approved for human use in 1985) and had demonstrated activity against a wide-range of microbes (Debnath et al., 2012; Jackson-Rosario et al., 2009; Tejman-Yarden et al., 2013), investigators decided to examine its anti-Giardia activity in more detail. Follow-up studies with auranofin demonstrated that this gold containing compound could effectively inhibit the growth of assemblage A and B parasites (Tejman-Yarden et al., 2013). Auranofin was also shown to be effective at inhibiting the growth of multiple MTZ-resistant parasite lines (Tejman-Yarden et al., 2013). While 50% growth inhibitory concentrations were demonstrated to be reasonably high in these studies (4–6 μM (Tejman-Yarden et al., 2013)), available pharmacokinetic data suggested that these concentrations could be achieved in vivo and hence encouraged further studies (Gottlieb, 1986; Tejman-Yarden et al., 2013). As pre-clinical studies performed in mice and gerbils, were also supportive of further development, with data demonstrating the clearance of parasites at tolerable doses (5–10 mg/kg for 5 days) (Tejman-Yarden et al., 2013), auranofin has now progressed into clinical trials (Capparelli et al., 2017). A phase I clinical trial investigating the pharmacokinetics and safety of auranofin (6 mg) when administered daily for 7 days in healthy subjects has been completed (Capparelli et al., 2017). Data from this study and previous work suggest that this treatment regimen is well-tolerated and is likely to achieve effective concentrations (13 μM) (Capparelli et al., 2017). Importantly, side-effects were reported in 46.7% of the candidates receiving compound, however, these were considered mild and all resolved without intervention. These promising data, together with evidence that higher doses and longer treatment regimens can be toleratated, has meant that auranofin is now being investigated in phase IIa clinical trials for the treatment of giardiasis and additional gastro-intestinal protozoa. However, it must be recognized that the use of this drug to treat giardiasis is likely to be associated with limitations and additional consequences. Auronafin is contraindicated during pregnancy and is broadspectrum antimicrobial agent which may impact the general health of the human gut microbiota.

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The anti-Giardia actvity of auranofin is not completely understood. While studies have demonstrated that auranofin is a potent inhibitor of parasite TrxR (IC50 against recombinant enzyme 152 nM; 100 the activity seen against human TrxR (Tejman-Yarden et al., 2013)), overexpression of this enzyme does not change the senstivity of parasites to the drug (Leitsch et al., 2016). These data suggest that mutliple mechansims may be at play, however, further investigations are needed to examine this idea. A multifaceted mode of action would certainly help in preventing the development of drug resistant parasites going forward, however, the morphological consequences of auraonfin treatment have been shown to mimick those seen with a control TrxR inhibitor (Tejman-Yarden et al., 2013). Certainly, a clear understanding of the anti-Giardia action of auranofin will be important going forward, particularly given that it may be intricately linked to the activity of MTZ and other currently used anti-Giardia chemotherapeutics (Leitsch et al., 2016). As previously discussed, TrxR is a redox enzyme known to be involved in the activation or reduction of MTZ and other 5-nitroimidazole drugs (Leitsch et al., 2011). It is also a target of the reduced intermediates of 5-nitroimidazole drugs (Leitsch et al., 2011). 5.2.2 Novel chemotype library screening Phenotypic screens using compound libraries that do not necessarily contain known bioactive compounds or compounds approved for clinical use can increase chemical diversity and facilitate the discovery of compounds with novel biological activities and previously unexploited modes of action. However, the limited availability of biological data often associated with these libraries can mean that drug discovery efforts take longer and are more expensive than re-purposing strategies. In the Giardia drug discovery field very few research groups have investigated these types of libraries for novel inhibitors of Giardia parasites. Indeed, to the best of our knowledge, the only screen of this nature that has been performed has yet to be published (Skinner-Adams et al., 2018). However, it is encouraging that this work appears to have identified three novel anti-Giardia compounds series with potent and selective (IC50 values <1 μM with the most promising candidate reported to be 290-fold more potent than MTZ with a selectivity index of >9000) activity against assemblage A, B and MTZ resistant parasites (Skinner-Adams et al., 2018). Importantly pre-clinical studies in a mouse model of giardiasis with the most potent compound identified from this work has also demonstrated good tolerability and in vivo efficacy at low concentrations (75% efficacy; 0.7 mg/kg daily for 3 days) (Skinner-Adams et al., 2018). Additional studies

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49

are on-going and are of significant interest going forward, given the potency and potential novel mode of action of these compounds. The library used by researchers in this screen was the Compounds Australia Scaffold Library. This library was of particular interest as it was built to provide chemical diversity and novel lead-like compounds in scaffold sets which facilitates rapid SAR assessments (Simpson and Poulsen, 2014). The library (34,000 compounds) was also available in subsets of 1, 2 or 4 compounds from each scaffold, a factor which reduced screening costs while still facilitating the identification of novel anti-Giardia chemotypes. The group screened a subset of the library (2541 compounds; 2/scaffold; Z’ factor 0.75). Given the preliminary success of this screening strategy combined to additional screens of this nature, including unexplored natural product libraries, may well be a mechanism to identify new anti-Giardia treatments for the future.

5.3 Target function-based drug discovery As the genome of multiple Giardia parasite lines is now available (GiardiaDB. org) the rational synthesis of new chemical entities based on new targets is possible. Indeed, 149 drug targets have been proposed using genomic data (Morrison et al., 2007). However, only 16 of these targets are currently targeted by known anti-Giardia compounds (Table 4). As a mechanism to identify novel inhibitors with different modes of action against parasites, researchers are now beginning to exploit this knowledge, instigating target-based drug discovery approaches using protein and protein families with defined and important biological functions in Giardia parasites. 5.3.1 Anti-oxidant enzyme inhibitors G. duodenalis has a “non-conventional” anti-oxidant system that includes around 20 enzymes (Ansell et al., 2015; Mastronicola et al., 2016). In host-parasite interactions these proteins are up-regulated in to pro-oxidant products secreted by epithelial cells such as NO (Ma’ayeh et al., 2017); likewise ABZ-resistant clones derived from WB strain over express anti-oxidant enzymes including Pxr1a, NADHox and FDP (Arguello-Garcia et al., 2015). Despite the important role that these enzymes play in parasite survival, very few have been targeted by drug discovery programs. Genomic data suggest that TrxR is the most druggable anti-oxidant enzyme of Giardia (Table 4) and its disulphide reductase activity is inhibited by thiol-active drugs including NBDHEX and auranofin (Brogi et al., 2017; Leitsch et al., 2016). The thiol-modifying compound, allicin, has also been

Table 4 Drug targets identified in G. duodenalis (strain WB, GL50803) from 149 ones originally proposed in GiardiaDB. Score Value

Compound

References

112079 17986283 Tubulin, alpha 1a

Alpha tubulin

830

0

NBDHEX Oryzalin

Camerini et al. (2017), Terra et al. (2010)

136021 29788768 Tubulin, beta 2B

Beta tubulin

809

0

Benzimidazole carbamates

Morgan et al. (1993)

98054

20149594 heat shock 90kDa protein 1, beta

Heat shock protein HSP 90- alpha

404

1.00E-113

2-aminobenzamide derivatives

Debnath et al. (2014)

86444

20127541 Serum glucocorticoid regulated

Kinase, AGC PKA

266

4.10E-71

Hispidin

Bazan-Tejeda et al. (2007)

16034

24308326 BR serine/ threonine kinase

Kinase, CAMK CAMKL

254

4.10E-70

Bumped kinase inhibitors (BKIs)

Hennessey et al. (2016)

11364

48255885 Protein kinase C, iota

Kinase, AGC AKT (PKB)

227

3.10E-59

Bumped kinase inhibitors (BKIs)

Hennessey et al. (2016)

94582

4505489

Ornithine decarboxylase

223

3.10E-58

Difluoromethylornithine Gillin et al. (1984)

17368

24308123 Serine/threonine kinase 36, fused

Kinase, ULK

216

2.10E-55

Bumped kinase inhibitors (BKIs)

Hennessey et al. (2016)

14019

4503139

Cathepsin B precursor

171

1.10E-42

Allicin

Arguello-Garcia et al. (2018)

Query GI

Definition

Ornithine decarboxylase

Cathepsin B precursor (Giardipain-1)

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Annotation in GiardiaDB

ORF

38788445 Ornithine Ornithine 148 carbamoyltransferase carbamoyltransferase

1.10E-35

N-phosphonoacetylL-ornthine, NBDHEX

Galkin et al. (2009), Camerini et al. (2017)

17406

34761064 Phosphoinositide-3- PI3K, class 3 kinase, class 3

119

2.10E-26

LY294002

Cox et al. (2006)

9421

29029632 Anaplastic lymphoma kinase Ki-1

Kinase, NEK

83

3.10E-15

Bumped kinase inhibitors (BKIs)

Hennessey et al. (2016)

13215

18490991 T-LAK-cell originated protein

Kinase, NEK

80

4.10E-15

Bumped kinase inhibitors (BKIs)

Hennessey et al. (2016)

15897

40254422 Nitric oxide synthase 3

Hypothetical protein

80

2.10E-14

Isothiourea hydrohalides Kazimierczuk et al. (2010)

8350

4759226

Transforming growth factor, beta

Kinase, NEK

75

2.10E-13

Bumped kinase inhibitors (BKIs)

Hennessey et al. (2016)

9827

21389617 Apoptosis-inducing factor like

Thioredoxin reductase

69

2.10E-11

NBDHEX Auranofin

Brogi et al. (2017), Camerini et al. (2017), Leitsch et al. (2016)

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10311

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Raúl Arg€ uello-García et al.

shown to inhibit TrxR (Ankri and Mirelman, 1999). PFOR, another 5-nitroheterocyclic drug activating enzyme, is inhibited by NTZ through a mechanism targeting the activated co-factor lactyl-thiamine pyrophosphate but not the substrate or catalytic sites (Hoffman et al., 2007). NTZ and its deacetylated metabolite tizoxanide, but not the secondary metabolite tizoxanide glucuronide inhibit the activity of NR1, another nitro-reducing enzyme (Muller et al., 2007b). Using bioinformatics approaches it has been suggested that tiliroside, a glyconated flavonoid from the medicinal plant Sphaeralcea angustifolia used to treat diarrhoea and dysentery in Mexico, interacts with giardial PFOR with similar affinity and inhibition energies than MTZ (Calzada et al., 2017). Another antioxidant giardial enzyme, the alcohol dehydrogenase E, has been proposed as target of cyclopropyl and cyclobutyl carbinols on the basis of its striking similarity to its E. histolytica homologue that is effectively inhibited by these compounds (Espinoza et al., 2004) As a whole, these observations support the incorporation of anti-oxidant enzymes of this parasite in future screenings of drug libraries to define their potential as drug targets in Giardia that can be useful for the treatment of giardiasis. 5.3.2 Bumped kinase inhibitors (BKIs) Protein kinases of Giardia are an attractive but an as yet unexploited class of drug targets. The striking sequence and structural divergence between giardial and human kinases (averaging 40% sequence identity), makes the 278 protein kinases and the 198 NEK (never-in-mitosis-related) kinases of Giardia promising candidates for drug development. Indeed the NEK kinases found in these parasites have only 1 yeast and 11 human homologues (Hennessey et al., 2016). In the search for calcium-dependent protein kinase (CDPK) inhibitors in Toxoplasma gondii and Cryptosporidium parvum, an in-house library of 36 compounds with different scaffolds was obtained ( Johnson et al., 2012). Interestingly, these compounds were directed to the atypically small gatekeeper residue (threonine and smaller as alanine, glycine and serine) located at the ATP binding pocket of CDPKs. These molecules, known as bumped kinase inhibitors (BKIs), may access the ATP binding pocket that is enlarged in these kinases by the reduced sized of side chain in the gatekeeper residues mentioned, which do not clash with BKIs, thus competing with ATP. Bioinformatics mining of GiardiaDB identified an initial set of putative BKI targets with threonine (5), glycine (2), serine (1) and alanine (1) in inactive pockets as gatekeeper residues. These active kinases include gULK-type

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53

kinase, gCAMPK/AMPK kinase, gPKB/AKT (Kim et al., 2005) and a host of NEK kinases (Hennessey et al., 2016). A phenotypic in vitro screen of 36 BKIs (tested at 5 μM) against Giardia trophozoites yielded five candidates that reduced parasite growth by >50%. In a further screen examining binding affinity of 400 BKIs towards purified NEK (GL50803_8445) and gCAMK two kinases critical for cytokinesis and attachment, using a thermal shift assay, compound 1264 was identified as a binder of gCAMK (ΔTm > 5) and compound 1213, demonstrated intermediate affinity to both proteins (ΔTm
2–3). Importantly both compounds were also shown to inhibit the in vitro growth of trophozoites (IC50 ¼ 0.9 and 0.8 μM, respectively). Compound 1213 was also effective against MTZsensitive clone 713 (IC50 ¼ 1.2 μM) and MTZ-resistant clone 713-M3 (IC50 ¼ 0.6 μM) (Hennessey et al., 2016). These BKI studies, support further work investigating the potential of such inhibitors as alternative treatments for giardiasis. Additional studies examining the activity of NEK candidates, present in other drug libraries, are particularly important given the action of these compounds appears to be very specific (BKIs that interact with only 6 of 15 identified drug targets in GiardiaDB have currently been identified) (Table 4). This family of molecules is particularly interesting going forward as they target proteins that are not targeted by current. 5.3.3 Inhibitors of virulence factors As virulence factors are central to the pathogenesis of Giardia, they represent potential targets for drug development. These factors are produced by trophozoites to facilitate multiple processes including; (a) intestinal colonization, (b) evasion of the host immune response, and (c) the acquisition of the nutrients for its growth. These molecules have been identified mostly by transcriptomic and proteomic methodologies in trophozoite-epithelial co-cultures and are cytoplasmic, localized at specific organelles and structures or even secreted to the extracellular milieu (Ma’ayeh et al., 2017). Virulence factors can be categorized as catalytic proteins, metabolic enzymes, surface molecules and soluble mediators (revised by OrtegaPierres and Arg€ uello-Garcı´a, 2019). Currently, the study of the precise role and the underlying pathogenic mechanisms of most of these components are still under progress. VSPs, high cysteine membrane proteins (HCMPs) and the structural/ contractile proteins within the genus-specific ventral disc of Giardia trophozoites (i.e. tubulins and giardins), are crucial to trophozoite host gut

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colonization. Inhibitors of these proteins have been identified and inhibit parasite development. For example, α-tubulin is targeted by the repurposed anti-cancer agent NBDHEX (Camerini et al., 2017), and by the dinitroaniline herbicide Oryzalin (Terra et al., 2010); likewise β-tubulin is a target of the benzimidazole carbamates (MacDonald et al., 2004; Morgan et al., 1993) (Table 4). All of these compounds inhibit cytokinesis, however, ABZ and other benzimidazoles cause ventral disc disassembly, rapid detachment and morphological distortion of trophozoites, while oryzalin provokes flagella retraction without effect on ventral disc (Morgan et al., 1993; Terra et al., 2010). VSPs also play an important role in host immune response evasion (Nash, 1997; Singer et al., 2001). In addition, parasites secrete proteases of the cysteine-type (mainly GL50803_14019 or Giardipain-1, GL50803_16160 and GL50803_16779 or CP3) that disrupt mucins, villin, intercellular junction proteins, immunoglobulins, defensins and cytokines (Liu et al., 2018, 2019). To date, besides disulfiram which was originally screened as a zinc finger-active compound (Nash and Rice, 1998), no other specific ligands or inhibitors of validated VSPS (
270 members) have been identified. In the case of cysteine proteases, the anti-fungal epoxide E-64 and its synthetic analogue CA-074 have been shown to block their activity. Recent studies have also demonstrated that the garlic derivative, allicin, a diallyl thiosulfinate generated by the action of the enzyme alliinase and its substrate alliin (S-allyl-L-cysteine sulfoxide), inhibits the proteolytic activity of Giardipain-1 and some other cathepsins B and cysteine proteases in G. duodenalis trophozoites leading to parasite death. The intragastric administration of allicin was able to eliminate trophozoites in Mongolian gerbils. It is believed that thiol-disulphide conversion, which is involved in the cysteine thioallylation induced by allicin effects several cysteine-rich molecules including cysteine proteases, VSPs, and HCMPs, with data demonstrating that the inhibition of Giardipain-1 by allicin abolishes the damage induced by this protease which includes intestinal cell membrane blebbing, disruption of cell junctions, caspase-3 activation and phosphatidylserine exposure in outer cell surface (Arguello-Garcia et al., 2018; Ortega-Pierres et al., 2018). In relation to factors involved in nutrient acquisition by Giardia it is important to consider arginine, which is in high demand by both parasites and intestinal epithelial cells. Trophozoites utilize extracellular L-arginine as a source of ATP by the arginine dihydrolase pathway (ADHP), whereas epithelial cells convert L-arginine into nitric oxide (NO), an important

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innate immunity mediator with giardicidal (Eckmann et al., 2000; Stadelmann et al., 2013). Two enzymes of the ADHP have been detected in the trophozoite secretome: ornithine carbamoyltransferase (gOCT) and arginine deiminase (gADI) (Dubourg et al., 2018; Ma’ayeh et al., 2017). These enzymes play a major role in the depletion of arginine in the intestine which can inhibit intestinal cell proliferation (Stadelmann et al., 2012). Structure-based drug-design (SBDD) approached have been used to identify compounds targeting arginolytic enzymes (Ortega-Pierres and Arg€ uelloGarcı´a, 2019). In these assays, the published structures of gOCT (PDB ID: 3GRF) and carbamate kinase (gCK, PDB ID: 3KZF) have been used to identify potential inhibitors. Importantly the structure of gOCT in sufficiently different to its human homologue to consider the design of specific inhibitors (Galkin et al., 2010) and gCK has not homologues in the human genome. A functional screen of two drug libraries for gCK inhibitors, identified disulfiram as a highly effective gCK inhibitor (IC50 ¼ 0.58 μM) with attractive giardicidal potency (IC50 ¼ 0.90μM) (Galkin et al., 2014). Inhibitors of the multifunctional gADI that participates in the processing of VSPs, s have also been identified. These include L-canavanine, a L-arginine analogue that is naturally present in alfalfa and in leguminous seeds and S-nitroso-L-homocysteine. With an oxa group instead of a methylene bridge, L-canavanine reversibly inhibits gADI by modifying its active site Cys400 (Li et al., 2008). S-nitroso-L-homocysteine inhibits gADI by forming S-nitroso and N-thiosulfoximide adducts that irreversibly inactivate the enzyme (Li et al., 2009). While, the use of L-canavanine may be hampered by the appearance of lupus-like reactions in mice and humans (Akaogi et al., 2006; Montanaro and Bardana, 1991), these preliminary studies highlight the potential of cysteine-modifying compounds against virulence factors of Giardia as new strategy for drug development. Of importance in this regard is the large repertoire of these proteins in this protozoan. Indeed, research examining the potential of such inhibitors is growing in the Giardia field.

5.4 Natural product drug discovery Products or compounds naturally synthesized by organisms including bacteria, plants and animals can be a great source of anti-giardial agents. In this context natural products are increasingly being studied for their anti-Giardia effects. As an example, many plant-based extracts (mainly ethanol, methanol and n-butanol extracts of roots, bark, leaves, rhizomes and flowers) have been obtained throughout the world from species traditionally endowed

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with anti-diarrheal activities, and further evaluated against G. duodenalis trophozoites. While many new compounds with anti-Giardia effects have been identified, the underlying mechanisms of action have been rarely reported. A galacto-glycerolipid isolated from the weed creeping woodsorrel (Oxalis curniculata) that is used for the treatment of dysentery and diarrhoea has demonstrated to cytotoxicity against trophozoites. While effects include trophozoite detachment, membrane blebbing and cytolysis (Manna et al., 2010), the target(s) of this galacto-glycerolipid have not yet been defined. The Mexican traditional medicine Bursera fagaroides or “Iztac qhuauhxiotl” which has been used as an anti-inflammatory, anti-cancer and anti-diarrheal agent, has also displayed good potency against Giardia trophozoites (IC50 ¼ 2.12 μM). While data suggest that this compound interferes with tubulin dimerization (Gutierrez-Gutierrez et al., 2017, 2019), in consistency with the presence of acetylphyllotoxin as its main component, this remains to be conclusively demonstrated. However the selective toxicity of iztac qhuauhxiotl is poor (4.1 times lower toxicity against Caco2 cells). Similarly, aqueous garlic extracts (AGE) have been shown to kill trophozoites by compromising exo- and endo-membrane integrity. It is believed that the major component of AGE, diallyl thiosulfinate (allicin), inhibits multiple cysteine proteases as previously described (see Section 5.3.3) (Arguello-Garcia et al., 2018; Harris et al., 2000). However, additional components in these extracts may also be, at least in part, associated with its anti-Giardia activity. Among anti-parasitic phytochemicals of outstanding interest are the polyphenols that include families of secondary plant and fungi metabolites such as flavonoids, isoflavonoids and neoflavonoids. In a series of ten isoflavones isolated from Dalbergia frutescens bark, formononetin (7-hydroxy-3-(4-methoxyphenyl]chromen-4-one), which is also found in red cloves and beans, was shown to exhibit good potency against Giardia trophozoites (IC50 ¼ 0.1 μM). However, the close structural analogues formononetin, genistein and daidzein were 100-fold less active (Khan et al., 2000). Formononetin (0.235 mg/g) was also effective (>80% reduction in trophozoite load) in a mouse model of giardiasis (Lauwaet et al., 2010). While the mode of action of formononetin has not been investigated, affinity chromatography coupled to mass spectrometry has been used to identify a nucleoside hydrolase homologue (GL50803_13272) as the target of daidzein. A clone resistant to these polyphenols (C3) has also been generated. This clones was shown to be sensitive to MTZ. Transcript levels of GL50803_13272 were also significantly reduced in this clone, supporting previous mode of action studies (Sterk et al., 2007). In concurrent studies,

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the flavonoids kaempferol, tiliroside and ()-epicatechin, isolated from four Mexican medicinal plants, were shown to be active against experimental giardiasis in suckling mice (ED50 values of 2.057, 1.429 and 0.072 μmol/kg, respectively), the latter being more effective than MTZ (Barbosa et al., 2007). Other natural phenols produced in plants, known as chalcones, have been used as scaffolds for the synthesis and evaluation of new anti-giardial agents. From a library of 46 piperidine- and piperazine-based chalcones, two of these had IC50 values in the 12.3–21.0 μM range under anaerobiosis and in the 1.6–3.0 μM range under microaerobiosis with 5–15 times lower toxicity against Caco2 cells (Bahadur et al., 2014). The assessment of another 45 triazolyl-quinolone-based chalcones identified 18–45 compounds, depending on oxygen availability in assays, with better in vitro activity than MTZ (IC50 < 3.4 μM). Of note, four derivatives (named as 29, 30, 51 and 65) have selectivity indexes >100 when compared with CaCo2 cell sensitivity (Bahadur et al., 2015). In some pioneering studies crude garlic extracts (Allium sativum L.), diluted 1:20 with water, were administered to mice (100 mL two times a day for 5 days) to 26 Egyptian children with giardiasis (Soffar and Mokhtar, 1991). Data suggested this natural product to be safe and efficacious. Another spice used as medicinal plant in Eurasia, Anethum graveolens or “Dill”, was administrated as an aqueous extract (AGAE) to 14 Iraqi children with giardiasis, aged 3–11 months at a dose of 1 mL 3 times a day for 5 days. Analyses of stool samples indicated parasitological cure at 14 days post-treatment in all children as with MTZ, with significant reduction in peristalsis by 5 days post-treatment and AGAE was well tolerated (Sahib et al., 2014). From these studies, it is tempting to speculate that plant extracts could be applied with efficacy and safety to clinical infections of Giardia; however, there nature product extracts can cause harm. For instance, aqueous extracts of the leaves from Piper betle or “Betel”, a Piperaceae plant highly consumed at South and Southeast Asia, when given intragastrically at dose 40 mg/100 g two times a day for 10 days in Mongolian gerbils while effective in eliminating trophozoites (Peckova et al., 2018), resulted in the release of tannins that were suspected to be associated with adverse health effects and chromosome aberrations in human leukocytes (Mori et al., 1979). Indeed, rigorous toxicological analyses are needed in the preparation of extracts from natural resources before these can be used in the clinical practise. In other studies, some components of animal origin, mainly from milk, have been tested for anti-giardial activity. This includes lactoferrin (LF), an

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80 kDa protein present in human milk, saliva, nasal secretions and tears with intrinsic iron-transfer functions and anti-microbial activity. Data from these studies suggest that LF and its derived peptides are cytotoxic to Giardia trophozoites (Turchany et al., 1995). It appears that LF binds to the parasite surface in an iron-dependent manner, causing endocytosis, membrane and cytoskeleton alterations which culminate in an apoptotic-like death (Aguilar-Diaz et al., 2017; Frontera et al., 2018; Turchany et al., 1997). The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase has been identified as an intracellular LF-receptor (Rawat et al., 2012) but it remains to be defined if a similar receptor is present in Giardia. At the clinical level, LF supplementation (5 g twice a day for 9 months) resulted in a significantly lower incidence of giardiasis (9.6%) as compared to placebo group (17.2%, n ¼ 174) in 146 Peruvian children (Ochoa et al., 2008). These data are encouraging and support the future evaluation of LF as treatment and perhaps giardiasis prophylactic in paediatric populations. A natural product with mixed animals and plants components that has been evaluated for antigiardial activity is propolis, “propolisina” or “bee glue”. Propolis, a resinous hive product from worker bees has been observed to have anti-Giardia activity. In these studies propolis from three locations in the Sonoran desert, Mexico, were shown to have seasonal (summer > winter > spring > autumn) anti-Giardia activity, with some components, including caffeic acid phenethyl ester, naringenin, hesperetin and pinocembrin found to be effective against trophozoites in the 222–680 μM range (Alday-Provencio et al., 2015). In a Cuban study with 138 giardiasis patients, propolisina (10–20%) cured >50% of all patients after 5 treatment (Miyares et al., 1988). In experimental giardiasis of mice, propolis reduced trophozoite loads to the same extent as MTZ, but was associated with a high inflammatory response (Abdel-Fattah and Nada, 2007). These observations suggest that further studies investigating the anti-Giardia activity of propolis constituents is warranted addressed to identify active components for further examination as alternation giardiasis therapies. Probiotics are live microorganisms such as bacteria and fungi that upon consumption replicate in the gut, releasing CpG-rich DNA, metabolites (e.g. tryptophan- and histamine-related) and enzymes (lactase and bile salt hydrolases (BSHs) among others) (Lebeer et al., 2018) which restore gut flora, modulate immune responses and antagonize pathogen virulence. Probiotic microorganisms have also been investigated as a mechanism to control Giardia infection. Bifidobacterium longum 51A, Weissella mesenteroides and Saccharomyces boulardii, for example, have demonstrated prophylactic

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activity when administered to Mongolian gerbils prior to infection (Fonseca et al., 2019; Ribeiro et al., 2018). In mice, prophylactic treatment with Enterococcus faecium SF68 and Lactobacillus rhamnosus GG (LGG) reduced trophozoites burden by increasing intestinal IgA, serum IgG and Peyer patches CD4+ counts (Benyacoub et al., 2005; Goyal and Shukla, 2013). Lactobacillus casei, a classical probiotic species, is also prophylactic in normal and malnourished animals, restoring anthropometric and histological abnormalities (Shukla et al., 2008, 2012). Interestingly, studies examining the role of probiotics protein products in mediating the anti-Giardia activity of specific probiotic organisms suggest that prophylactic activity of probiotics is mediated by proteins rather than the organisms themselves. As an example, total protein extracts from LGG were shown to be prophylactic, with treatments preserving intestinal epithelium and inducing IgA and NO production (Shukla et al., 2019). Further, BSHs were directly shown to be responsible for the anti-Giardia activity of probiotic L. johnsonii La1 (NCC533), which upon secretion, hydrolyze the amido group of glycine- and taurine-conjugated bile salts, generating free acids (cholic, deoxycholic and chenodeoxycholic) that are cytotoxic to trophozoites (Travers et al., 2016). Recombinant BSHs, namely rBSH47 and rBSH56, were shown to be active against trophozoites in vitro in the presence of bile and rBSH47 (50 μg/day for 5 days) was shown to reduced trophozoite counts (69%) in the intestines of infected suckling mice (Allain et al., 2018b). Additional evidence included the demonstration that the anti-Giardia activity of 29 different Lactobacillus strains was directly associated with their expression of BSHs (Allain et al., 2018a). In conclusion, while all of the currently data suggest that the use of probiotic bacteria and fungi is useful for Giardia prophylaxis, the benefits of these treatments to patients or animals with established infections required further study. In this context, a study in which dogs chronically infected with Giardia were treated with E. faecium SF68 did not show effects on immune responses or parasite elimination (Simpson et al., 2009).

6. Conclusions and perspectives Over the past three decades six anti-giardial drugs have been available for the clinical treatment of giardiasis. However, increasing rates of therapeutic failures, particularly with our most commonly used agents, the 5-nitroimidazoles, are becoming a significant concern. To improve current treatment options, there is great interest in the identification of new

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anti-giardial agents. In this context, omic technologies are being used to identify new drug targets and to improve our understanding of resistance mechanisms, as with this information we are better placed to monitor resistance and to develop improved, alternative treatments. While we have come a long way in identifying new drug-targets and understanding some of the mechanisms associated with current drug resistance, there is still a long way to go. Although we now have genome data for both human infecting G. duodenalis assemblages and have identified some 150 potential drug targets, we have yet to exploit many of these as drugs. Current resistance mechanism studies have also been confined to in vitro selected parasite lines, which makes the clinical relevance of the data generated difficult to ascertain. This is a particularly true for 5-nitroimidazole resistance, given that current data are complex and suggest that multiple mechanisms and molecular markers are in play. Further studies examining resistance in clinical isolates are desperately needed to ascertain whether the same diversity is seen in the field. In the context of target-guided drug discovery, researchers have begun to look at specific drug targets including antioxidant enzymes (TrxR, PFOR, NR1 and AdhE), bumped kinases (ULK-type, PKB and NEK kinases) and virulence factors (cysteine proteases as Giardipain-1 and arginolytic enzymes as carbamate kinase and arginine deiminase). Some of these studies are identifying promising leads. However, large protein families such as VSPs and HCMPs are yet to be explored as drug targets. Research examining the anti-Giardia activity of known analogues with activity against resistant parasites is also ongoing, with research in this area identifying some exciting new potent compounds. This includes multiple next generation nitroimidazoles (>500) and benzimidazoles (10) with more potent in vitro activity than MTZ and ABZ, respectively. However, arguably the most promising advances have been derived from phenotypic screens, particularly those that have focused on libraries of known biological active compounds and drugs. These studies have identified novel anti-Giardia agents (i.e. orlistat, NBDHEX, auranofin, omeprazole, disulfiram and propranolol) with potent activity, one of which, auranofin has progressed to phase II clinical trials. Further phenotypic studies have also identified a number of natural products and new synthetic compounds with potent activity against parasites. While the in vivo potential of many of these compounds is yet to be ascertained, the anti-Giardia drug discovery pipeline is beginning to look healthier. As new drug candidates progress in this pipeline, mechanisms of drug deployment, including the use of combination treatment strategies, should be

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considered to prevent the development of further drug resistance. For instance, it is still tempting to evaluate the efficacy of dyadic combinations of drugs of current use (e.g. nitroheterocycles and benzimidazoles). Finally, the information presented here provides a comprehensive status of the art regarding chemotherapy failures in giardiasis and G. duodenalis resistance mechanisms together with advances in alternative strategies for future evaluations of compounds from different sources. Other novel strategies such as the use of controlled drug delivery systems as nanoparticles and dendrimers (Mhlwatika and Aderibigbe, 2018; Said et al., 2012) would shortly afford additional tools to optimize efficacy of current and future anti-giardial agents of clinical and veterinary prescription.

Acknowledgements We are grateful to Arturo Perez-Taylor for informatics support. We would like to acknowledge financial support from the Miguel Alema´n Foundation and Cinvestav, SEPCinvestav consortium and the Australian National Health and Medical Research Council (TSA APP1141069).

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